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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization

International Bureau

(43) International Publication Date
27 December 2001 (27.12.2001)

(10) International Publication Number

PCT WO 01/97858 A2

(51) International Patent Classification 7 : A61K 51/00 (81) Designated States (national): AE, AG, AL, AM, AT, AU,

(21) International Application Number: PCT/US0 1/1 8939

(22) International Filing Date: 14 June 2001 (14.06.2001)
(25) Filing Language: English

(26) Publication Language:

(30) Priority Data:

60/212,668

20 June 2000 (20.06.2000) US

(71) Applicant: IDEC PHARMACEUTICALS CORPORA-
TION [US/US]; 3030 Callan Road, San Diego, CA 92121
(US).

(72) Inventor: WHITE, Christine; P.O. Box 9242, Rancho
Santa Fe, CA 92067 (US).

(74) Agents: TESKIN, Robin, L. et ai.; Pillsbury Winthrop
LLP, 1600 Tysons Boulevard, McLean, VA 22102 (US).

AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW.

English Designated States (regional): ARIPO patent (GH, GM,

KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).

Published:

— without international search report and to be republished
upon receipt of that report

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.

id — ■

00 (54) Title: TREATMENT OF B-CELL ASSOCIATED DISEASES SUCH AS MALIGNANCIES AND AUTOIMMUNE DIS-

^ EASES USING A COLD ANTI-CD20 ANTIBODY/RADIOLABELED ANTI-CD22 ANTIBODY COMBINATION
ON

(57) Abstract: Treatment of B-cell associated diseases including autoimmune and B-cell malignancies such as leukemias, lym-
phomas, using the combination of an anli-CD20 antibody, preferably RITUXAN® and a radiolabeled anti-CD22 antibody, prefer-

Q ably an "Y labeled humanized anti-CD22 antibody, is described. These therapeutic regimens provide for enhanced depletion of B
cells, and therefore reduce the risk in B cell malignancy treatment of relapse associated with RITUXAN® and, moreover, provide

^ for prolonged immunosuppression of B-cell immune responses, especially in the context of autoimmune diseases and transplant

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TREATMENT OF B-CELL ASSOCIATED DISEASES SUCH AS
MALIGNANCIES AND AUTOIMMUNE DISEASES USING A COLD
ANTI-CD20 ANTD3QDY/RADIOLABELED ANTI-CD22
ANTIBODY COMBINATION

Cross Reference to Related Application

This application claims priority from U.S. Provisional Serial No. 60/212,668,
filed June 20, 2000, and which is incorporated herein in its entirety by reference.

10 Field of the Invention

The present invention is concerned with a combination immunotherapy/-
radiotherapy involving the administration of a cold anti-CD20 antibody, preferably
RITUXAN®, or another anti-CD20 antibody having substantially the same B-cell
depleting activity as RITUXAN®, and a radiolabeled anti-CD22 antibody, preferably
15 an yttrium labeled humanized anti-CD22 antibody. In the case of tumor therapy, the
initial administration of the cold anti-CD23 antibody helps remove B cells from the
circulation, thereby improving the targeting and efficacy of the radiolabeled anti-
CD22 antibody.

Also, the subject treatment provides for enhanced immunosuppression vis-a-
20 vis cold CD20 and radiolabeled anti-CD22 therapy alone. This combination

therapeutic regimen is useful in the treatment of diseases wherein depletion and/or
selective killing, and/or blocking the function of CD20 and CD22 expressing cells is
therapeutically beneficial, especially B-cell malignancies, lymphomas, leukemias, and
conditions or diseases wherein suppression of B-cell immune function is .
25 therapeutically beneficial, e.g., autoimmune diseases, allergic diseases, transplant, and
other therapeutic regimens involving administration of antigenic moieties, e.g.,

protein, cell or gene therapy. Preferably, the therapeutic regimen will comprise the
initial administration of RITUXAN®,' followed by admhiistration of the radiolabeled
anti-CD22 antibody.

Background of the Invention
I. Anti-CD20 Antibodies

CD20 is a cell surface antigen expressed on more than 90% of B-cell
lymphomas and does not shed or modulate in the neoplastic cells (McLaughlin et al.,

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J. Clin. Oncol. 16: 2825-2833 (1998)). Anti-CD20 antibodies have been prepared for
use both in research and therapeutics. One reported anti-CD20 antibody is the
monoclonal Bl antibody (U.S. Patent No. 5,843,398). Anti-CD20 antibodies have
also been prepared in the form of radionuclides for treating B-cell lymphoma (e.g. ,
5 131 I-labeled anti-CD20 antibody), as well as a 89 Sr-labeled form for the palliation of
bone pain caused by prostate and breast cancer metastasises (Endo, Gan To Kagaku
Ryoho 26: 744-748 (1999)).

A murine monoclonal antibody, 1F5, (an anti-CD20 antibody) was reportedly
adrninistered by continuous intravenous infusion to B cell lymphoma patients.

10 However, extremely high levels (>2 grams) of 1F5 were reportedly required to deplete
circulating tumor cells, and the results were described as "transient" (Press et at,
Blood 69: 584-591 (1987)). A potential problem with using monoclonal antibodies
as therapeutics is that non-human monoclonal antibodies (e.g., murine monoclonal
antibodies) typically lack human effector functionality, e.g., they are unable to, inter

1 5 alia, mediate complement dependent lysis or lyse human target cells through
antibody-dependent cellular toxicity or Fc-receptor mediated phagocytosis.
Furthermore, non-human monoclonal antibodies can be recognized by the human host
as a foreign protein; therefore, repeated injections of such foreign antibodies can lead
to the induction of immune responses leading to harmful hypersensitivity reactions.

20 For murine-based monoclonal antibodies, this is often referred to as a Human

Anti-Mouse Antibody response, or "HAMA" response. Additionally, these "foreign"
antibodies can be attacked by the immune system of the host such that they are, in
effect, neutralized before they reach their target site.
A. RITUXIMAB®

25 RITUXIMAB® (also known as RITUXAN®, MabThera® and IDEC-C2B8)

was the first FDA-approved monoclonal antibody and was developed at DEC
Pharmaceuticals (see U.S. Patent Nos. 5,843,439; 5,776,456 and 5,736,137).
RITUXIMAB® is a chimeric, anti-CD20 monoclonal (MAb) recommended for
treatment of patients with low-grade or follicular B-cell non-Hodgkin's lymphoma

30 (Mclaughlin et al, Oncology (Huntingt) 12: 1763-1777 (1998); Leget et al, Curr.
Opin. Oncol. 10: 548-551 (1998)). m Europe, RITUXIMAB® has been approved for
therapy of relapsed stage IMV follicular lymphoma (White et al, Pharm. Sci.

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Technol. Today 2: 95-101 (1999)). Other disorders treatable with. RTTUXMAB®
include follicular centre cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse
large cell lymphoma (DLCL), and small lymphocytic lymphoma/chronic lymphocytic
leukemia (SLL/CLL) (Nguyen et al, 1999)). RITUXMAB® has exhibited minimal
5 toxicity and significant therapeutic activity in low-grade non-Hodgkin's lymphomas
(NHL) in phase I and II clinical studies (Berinstein et al.,Ann. Oncol. 9: 995-1001
(1998)).

RITUXMAB®, which is currently being used alone to treat B-cell NHL at
weekly doses of typically 375 mg/M 2 for four weeks with relapsed or refractory low-

1 0 grade or follicular NHL. This antibody is well tolerated and had significant clinical
activity (Piro etal.,Ann. Oncol. 10: 655-61 (1999); Nguyen et al, Eur. J. Haematol
62: 76-82 (1999); and Coiffier et al, Blood 92: 1927-1932 (1998)). Also, up to 500
mg/M 2 of four weekly doses have also been administered during trials using the
antibody (Maloney et al, Blood 90: 2188-2195 (1997)). RITUXIMAB® also has

1 5 been combined with chemotherapeutics, such as CHOP (e.g. , cyclophosphamide,

doxorubicin, vincristine and prednisone), to treat patients with low-grade or follicular
B-cell non-Hodgkin's lymphoma (Czuczman et al, J. Clin. Oncol. 17: 268-76 (1999);
and McLaughlin et al, Oncology (Huntingt) 12: 1763-1777 (1998)). However, it has
not previously been utilized in combination with other therapeutic antibodies.

20 The synthesis of monoclonal antibodies against CD22 and their use in

therapeutic regimens has also been reported. CD22 is a B-cell-specific molecule
involved in B-cell adhesion that may function in homotypic or heterotypic interactions
(Stamenkovic et al, Nature 344:74 (1990); Wilson et al, J. Exp. Med. 173:137 (1991);
Stamenkovic et al, Cell 66:1 133 (1991)). The CD22 protein is expressed in the

25 cytoplasm of progenitor B and pre-B-cells (Dorken et al, J. Immunol. 136:4470
(1986); Dorken et al, "Expression of cytoplasmic CD22 in B-cell ontogeny. In
Leukocyte Typing m, White Cell Differentiation Antigens. McMichael et al, eds.,
Oxford University Press, Oxford, p. 474 (1987); Schwarting et al, Blood 65:974
(1985); Mason et al, Blood 69:836 (1987)), but is found only on the surface of mature

30 B-cells, being present at the same time as surface IgD (Dorken et al, J. Immunol.
136:4470 (1986)). CD22 expression increases following activation and disappears
with further differentiation (Wilson et al, J. Exp. Med. 1 73 : 1 37 (1 99 1); Dorken et al,

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J. Immunol. 136:4470 (1986)). In lymphoid tissues, CD22 is expressed by follicular
mantle and marginal zone B-cells but only weakly by genninal center B-cells (Dorken
et al, Immunol. 136:4470 (1986); Ling et al, "B-cell and plasma antigens: new and
previously defined clusters" In Leukocyte Typing HI. White Cell Differentiation
5 Antigens, McMichael et al, eds., Oxford University Press, Oxford, p. 302 (1987)).
However, in situ hybridization reveals the strongest expression of CD22 mRNA
within the germinal center and weaker expression within the mantle zone (Wilson et
d,J.Exp. Med. 173:137 (1991)). CD22 is speculated to be involved in the regulation
of B-cell activation since the binding of CD22 mAb to B-cells in vitro has been found

10 to augment both the increase in intracellular free calcium and the proliferation induced
after cross-linking of surface Ig (Pezzutto et al, J. Immunol. 138:98 (1987); Pezzutto
et al, J. Immunol. 140:1791 (1988)). Other studies have determined, however, that the
augmentation of anti-Ig induced proliferation is modest (Dorken et al, J. Immunol.
1 36:4470 (1986)). CD22 is constitutively phosphorylated, but the level of

15 phosphorylation is augmented after treatment of cells with PMA (Boue et al, J.

Immunol. 140:192(1988)). Furthermore, a soluble form of CD22 inhibits the CD3-
mediated activation of human T-cells, suggesting CD22 may be important in T-cell -
B-cell interactions (Stamenkovic et al, Cell 66:1 133 (1991)).

Ligands that specifically bind the CD22 receptor have been reported to have

20 potential application in the treatment of various diseases, especially B-cell lymphomas
and autoimmune diseases. In particular, the use of labeled and non-labeled anti-CD22
antibodies for treatment of such diseases has been reported.

For example, Tedder et al, U.S. patent 5,484,892, that purportedly bind CD22
with high affinity and block the interaction of CD22 with other ligands. These

25 monoclonal antibodies are disclosed to be useful in treating autoimmune diseases
such as glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis,
lymphadenitis, periarteritis nodosa, systemic lupus erythematosis, arthritis,
thrombocytopenia purpura, agranulocytosis, autoimmune hemolytic anemias, and for
inhibiting immune reactions against foreign antigens such as fetal antigens during

30 pregnancy, myasthenia gravis, insulin-resistant diabetes, Graves' disease and allergic
responses.

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Also, Leung et al, U. S. Patent 5,789,557, disclose chimeric and humanized
anti-CD22 monoclonal antibodies produced by CDR grafting and tbe use thereof in
conjugated and unconjugated form for therapy and diagnosis of B-cell lymphomas and
leukemias. The reference discloses especially such antibodies conjugated to cytotoxic
5 agents, such as chemotherapeutic drugs, toxins, heavy metals and radionuclides. (See
U.S! Patent 5,789,554, issued August 4, 1998, to Leung et al, and assigned to
Immunomedics.)

Further, PCT applications WO 98/42378, WO 00/20864, and WO 98/41641
describe monoclonal antibodies, conjugates and fragments specific to GD22 and

1 0 therapeutic use thereof, especially for treating B-cell related diseases.

Also, the use of anti-CD22 antibodies for treatment of autoimmune diseases
and cancer has been suggested. See, e.g., U.S. Patent 5,443,953, issued August 22,
1995 to Hansen et al and assigned to Immunomedics Inc. that purports to describe
anti-CD22 immunoconjugates for diagnosis and therapy, especially for treatment of

15 viral and bacterial infectious diseases, cardiovascular disease, autoimmune diseases,
and cancer, and U.S. Patent 5,484,892, issued January 16, 1998 to Tedder et al and
assigned to Dana-Farber Cancer institute, Inc. that purports to describe various
monoclonal antibodies directed against CD22, for treatment of diseases wherein
retardation or blocking of CD22 adhesive function is therapeutically beneficial,

20 particularly autoimmune diseases.) These references suggest that an anti-CD22
antibody of fragment may be directly or indirectly conjugated to a desired effector
moiety, e.g., a label that may be detected, such as an enzyme, fluorophore,
radionuclide, electron transfer agent during an in vitro immunoassay or in vivo
imaging, or a therapeutic effector moiety, e.g., a toxin, drug or radioisotope.

25 Further, an anti-human CD22 monoclonal antibody of the IgGl isotype is

commercially available from Leinco Technologies, and reportedly is useful for
treatment of B-cell lymphomas and leukemias, including hairy cell leukemia.
(Campana, D. et al, J. Immunol. 134:1524 (1985)). Still further, Dorken et al, J.
Immunol. 150:4719 (1993) and Engel et al, J. Immunol. 150:4519 (1993) both

30 describe monoclonal antibodies specific to CD22.

Also, the combined administration of an anti-CD22 immunotoxin and an anti-
CD 1 9 immunotoxin has been reported for the treatment of diseases including cancer

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and autoimmune diseases. (See U.S. Patent 5,686,072, issued November 11, 1997 to
Uhr et al land assigned to The University of Texas.)

Therefore, based on the foregoing, while RTTUXAN® and other therapies
have been reported for treatment of B-cell lymphomas, often such treatments are
5 subject to relapse. Therefore, notwithstanding what has been reported relating to the
use of anti-CD20 antibodies and anti-CD22 antibodies in therapeutic regimens, it
would be an advantage if novel therapeutic regimens could be developed, especially
combination therapies that provide for enhanced therapeutic efficacy. In particular, it
would be advantageous if novel therapies could be developed that prevent or reduce
10 disease relapse in patients treated with RTTUXAN® or other anti-CD20 antibody
therapeutic regimens.

Preferred Embodiments of the Invention

It is an embodiment of the invention to provide a novel therapeutic regimen
1 5 that comprises the administration of an anti-CD20 monoclonal antibody or fragment
thereof, and the administration of a radiolabeled anti-CD22 monoclonal antibody or
fragment.

It is another embodiment of the invention to provide a novel therapeutic
regimen involving the initial administration of RTTUXAN®, followed by the
20 administration of a radiolabeled anti-CD22 monoclonal antibody, or fragment thereof.
It is another embodiment of the invention to provide a novel therapeutic
regimen for the treatment of B-cell malignancies and cancers, especially B-cell
leukemias or lymphomas, comprising the administration of RITUXAN®, followed by
a radiolabeled humanized anti-CD22 antibody.
25 It is another embodiment of the invention to provide novel methods for the

treatment of autoimmune diseases and transplant comprising the administration of
RITUXAN®, followed by a radiolabeled anti-CD22 antibody.

It is another embodiment of the invention to provide novel methods of
inhibiting B-cell immune responses, especially in protein, gene or cell therapy, or in
30 the treatment of allergic disorders by the combined ao!mimstration of an anti-CD20
antibody and a radiolabeled anti-CD22 antibody.

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Summary of the Invention

The present invention relates to methods of treating patients having diseases
wherein inhibiting and/or depleting and/or killing and/or blocking the formation of B-
cells is therapeutically desirable, especially B-cell malignancies and leukemias, as
5 well as autoimmune diseases, transplant, allergic disorders, inflammatory disorders
and gene or cell therapy, and other conditions wherein B-cell immunity is desirably
suppressed, ha a preferred embodiment, the present invention relates to the treatment
of B-cell lymphomas and leukemias, especially non-Hodgkin's lymphoma (NHL).
Essentially, the subject therapeutic regimen will comprise the admimstration
10 of a cold anti-CD20 antibody or fragment, and a hot (radiolabeled) anti-CD22
antibody or fragment. The anti-CD20 antibody and the radiolabeled anti-CD22
antibody can be administered in combination or separately, and in either order.
Preferably, the anti-CD20 antibody will be administered first, in sufficient amounts to
effect B-cell depletion, followed by the administration of a radiolabeled anti-CD22
15 antibpdy.

Preferably, this combination will affect synergistic results vis-a-vis the use of
the cold anti-CD20 or radiolabeled anti-CD22 antibody or fragment alone. In a
particularly preferred embodiment, this combination will provide for enhanced killing
or depletion of tumorigenic B cells because the cold anti-CD20 antibody initially
20 clears most CD20 expressing cells and the radiolabeled anti-CD22 antibody clears
substantially all remaining tumorigenic B cells. Optionally, the combination therapy
may further include the use of a radiolabeled anti-CD20 antibody, e.g. radiolabeled
2B8 (Zevalin®).

In another preferred embodiment, this combination will prevent or inhibit
25 relapse in patients with B cell malignancies, e.g. non-Hodgkin's lymphoma, vis-a-vis
current Rituxan®-based therapeutic regimens.

Detailed Description of the Invention

In order to clearly describe the invention, the following definitions are
30 provided.

WO 01/97858 PCT/US01/18939
Definitions

Units, prefixes, and symbols can be denoted in their Si accepted form.
Numeric ranges are inclusive of the numbers defining the range. Unless otherwise
indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid
5 sequences are written left to right in amino to carboxy orientation. The headings
provided herein are not limitations of the various aspects or embodiments of the
invention which can be had by reference to the specification as a whole. Accordingly,
the terms defined immediately below are more fully defined by reference to the
specification in its entirety.

10 The term "antibody" as used herein is intended to include immunoglobulins

and fragments thereof which are specifically reactive to the designated protein or
peptide thereof. An antibody can include human antibodies, primatized antibodies,
chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to
other proteins or radiolabels, and antibody fragments.

15 The term "antibody" herein is used in the broadest sense and specifically

covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies
(e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody
fragments so long as they exhibit the desired biological activity.

"Antibody fragments" comprise a portion of an intact antibody, preferably

20 comprising the antigen-binding or variable region thereof. Examples of antibody

fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies formed from antibody
fragments. Antibody fragments may be isolated using conventional techniques. For
example, F(ab : )2 fragments can be generated by treating antibodies with pepsin. The

25 resulting F(ab ! )2 fragment can be treated to reduce disulfide bridges to produce Fab 1
fragments.

"Native antibodies" are usually heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light (L) chains and two identical heavy
(H) chains. Each light chain is linked to a heavy chain by one covalent disulfide
30 bond, while the number of disulfide linkages varies among the heavy chains of
different immunoglobulin isotypes. Each heavy and light chain also has regularly
spaced intrachain disulfide bridges. Each heavy chain has at one end a variable

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domain (VH) followed by a number of constant domains. Each light chain has a
variable domain at one end (VL) and a constant domain at its other end; the constant
domain of the light chain is aligned with the first constant domain of the heavy chain,
and the light-chain variable domain is aligned with the variable domain of the heavy
5 chain. Particular amino acid residues are believed to form an interface between the
light chain and heavy chain variable domains.

The term "variable" refers to the fact that certain portions of the variable
domains differ extensively in sequence among antibodies and are used in the binding
and specificity of each particular antibody for its particular antigen. However, the

10 variability is not evenly distributed throughout the variable domains of antibodies. It
is concentrated in three segments called hypervariable regions both in the light chain
and the heavy chain variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable domains of native
heavy and light chains each comprise four FRs, largely adopting a 13-sheet

1 5 configuration, connected by three hypervariable regions, which form loops
connecting, and in some cases forming part of, the B -sheet structure. The
hypervariable regions in each chain are held together in close proximity by the FRs
and, with the hypervariable regions from the other chain, contribute to the formation
of the antigen-binding site of antibodies (see Kabat et al, Sequences of Proteins of

20 Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health,
Bethesda, MD. (1 99 1)). The constant domains are not involved directly in binding an
antibody to an antigen, but exhibit various effector functions, such as participation of
the antibody in antibody dependent cellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-binding

25 fragments, called "Fab" fragments, each with a single antigen-binding site, and a
residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin
treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still
capable of cross-linking antigen.

"Fv" is the minimum antibody fragment which contains a complete antigen-

30 recognition and antigen-binding site. This region consists of a dimer of one heavy
chain and one light chain variable domain in tight, non-covalent association. It is in
this configuration that the three hypervariable regions of each variable domain interact

WO 01/97858 PCT/US01/18939

to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the
six hypervariable regions confer antigen-binding specificity to the antibody. However,
even a single variable domain (or half of an Fv comprising only three hypervariable
regions specific for an antigen) has the ability to recognize and bind antigen, although
5 at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy terminus of the heavy chain
CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH

10 is the designation herein for Fab' in which the cysteine residue(s) of the constant

domains bear at least one free thiol group. F(ab')Z antibody fragments originally were
produced as pairs of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate

15 species can be assigned to one of two clearly distinct types, called kappa and lambda,
based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy
chains, antibodies can be assigned to different classes. There are five major classes of
intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further

20 divided into subclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgA, and IgA2. The
heavy-chain constant domains that correspond to the different classes of antibodies
are called alpha, delta, epsilon, gamma and mu, respectively. Preferably, the
heavy-chain constant domains will complete the gamma- 1, gamma-2, gamma-3 and
gamma-4 constant region. Preferably, these constant domains will also comprise

25 modifications to enhance antibody stability such as the P and E modification disclosed
in U.S. Patent No. 6,01 1,138 incorporated by reference in its entirety herein. The
subunit structures and three dimensional configurations of different classes of
immunoglobulins are well known.

"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL

30 domains of antibody, wherein these domains are present in a single polypeptide chain.
Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH
and VL domains which enables the scFv to form the desired structure for antigen

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binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.
269-315 (1994).

The term "diabodies" refers to small antibody fragments with two
5 antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH)
connected to a light chain variable domain (VL) in the same polypeptide chain (VH -
VL). By using a linker that is too short to allow pairing between the two domains on
the same chain, the domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites. Diabodies are described more fully

10 in, for example, EP 404,097; WO 93/1 1161; and Hollinger et al, Proc. Natl. Acad.
Sci. USA, 90:6444-6448 (1993).

The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for possible naturally

15 occurring mutations that may be present in rninor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site. Furthermore, in contrast
to conventional (polyclonal) antibody preparations which typically include different
antibodies directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the antigen, hi addition to their

20 specificity, the monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncxmtarninated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For example, the

25 monoclonal antibodies to be used in accordance with the present invention may be
made by the hybridoma method first described by Kohler et al, Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g.,\J.S. Patent No.
4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al, Nature, 3 52:624-628

30 (1991) and Marks et al, J. Mol Biol, 222:581-597 (1991), for example.

By "humanized antibody" is meant an antibody derived from a non-human
antibody, typically a murine antibody, that retains or substantially retains the antigen-

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binding properties of the parent antibody, but which is less immunogenic in humans.
This may be achieved by various methods, including (a) grafting the entire non-human
variable domains onto human constant regions to generate chimeric antibodies; (b)
grafting only the non-human complementarity determining regions (CDRs) into
5 human framework and constant regions with or without retention of critical

framework residues; and (c) transplanting the entire non-human variable domains, but
"cloaking" them with a human-like section by replacement of surface residues. Such
methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81 : 685 1-5 (1984);
Morrison et al, Adv. Immunol. 44: 65-92 (1988); Verhoeyen et al, Science 239:

10 1534-1536 (1988); Padlan, Molec. Immun. 28: 489-498 (1991); and Padlan, Molec.
Immun. 31: 169-217 (1994), all of which are hereby incorporated by reference in their
entirety. Humanized anti-CD40L antibodies can be prepared as described in U.S.
Patent Application No. 08/554,840 filed November 7, 1995 also incorporated herein
by reference in its entirety.

15 By "human antibody" is meant an antibody containing entirely human light

and heavy chain as well as constant regions, produced by any of the known standard
methods.

By "primatized antibody" is meant a recombinant antibody which has been
engineered to contain the variable heavy and light domains of a monkey (or other

20 primate) antibody, in particular, a cynomolgus monkey antibody, and which contains
human constant domain sequences, preferably the human immunoglobulin gamma 1
or gamma 4 constant domain (or PE variant). The preparation of such antibodies is
described in Newman et al, Biotechnology, 10: 1458-1460 (1992); also in commonly
assigned 08/379,072, 08/487,550, or 08/746,361, all of which are incorporated by

25 reference in their entirety herein. These antibodies have been reported to exhibit a
high degree of homology to human antibodies, i.e., 85-98%, display human effector
functions, have reduced immunogenicity, and may exhibit high affinity to human
antigens.

By "antibody fragment" is meant an fragment of an antibody such as Fab,
30 F(ab')2, Fab' and scFv.

By "chimeric antibody" is meant an antibody containing sequences derived
from two different antibodies, which typically are of different species. Most typically,

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chimeric antibodies comprise human and murine antibody fragments, and generally
human constant and murine variable regions.

"B Cell Depleting Antibody" therein is an antibody or fragment that upon
administration, results in demonstrable B cell depletion. Typically, such antibody will
5 bind to a B cell antigen or B cell marker expressed on the surface of a B cell.

Preferably, such antibody, after adrniriistration, typically within about several days or
less, will result in a depletion of B cell number by about 50% or more. In a preferred
embodiment, the B cell depleting antibody will be RITUXAN® (a chimeric anti-
CD20 antibody) or one having substantially the same or at least 20-50% the cell
1 0 depleting activity of RITUXAN®, over the same time period, preferably at least 90%
thereof.

A "B cell surface marker" or "B cell target" or "B cell antigen" is an antigen
expressed on the surface of a B cell which can be targeted with an antagonist which
binds thereto. Exemplary B cell surface markers include the CD10, CD19, CD20,

15 CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76,
CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and
CD86 leukocyte surface markers. A preferred B cell surface marker is preferentially
expressed on B cells compared to other non-B cell tissues of a mammal and may be
expressed on both precursor B cells and mature B cells.

20 The "CD20" antigen is a -35 kDa, non-glycosylated phosphoprotein found on

the surface of greater than 90% of B cells from peripheral blood or lymphoid organs.
CD20 is expressed during early pre-B cell development and remains until plasma cell
differentiation. CD20 is present on both normal B cells as well as malignant B cells.
Other names for CD20 in the literature include "B-lymphocyte-restricted antigen" and

25 "Bp35". The CD20 antigen is described in Clark et al. PNAS (USA) 82:1766(1985).

The "CD22" antigen refers to an antigen expressed on B cells, also known as
"BL-CAM" and "LybB" that is involved in B cell signaling and an adhesion. (See
Nitschke et al, Curr. Biol. 7:133 (1997); Stamenkovic et al, Nature 345:74 (1990)).
This antigen is a membrane immunoglobulin-associated antigen that is tyrosine

3 0 phosphorylated when membrane Ig is ligated. (Engel et al, J. Etyp. Med. 1 8 1 (4) : 1 52 1
1586 (1995)). The gene encoding this antigen has been cloned, and its lg domains
characterized.

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A B cell "antagonist" is a molecule which, upon binding to a B cell surface
marker, destroys or depletes B cells in a mammal and/or interferes with one or
more B cell functions, e.g. by reducing or preventing a humoral response elicited by
the B cell. The antagonist preferably is able to deplete B cells (i.e. reduce circulating
5 B cell levels) in a mammal treated therewith. Such depletion may be achieved via
various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC)
and/or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation
and/or induction of B cell death (e.g. via apoptosis). Antagonists included within the
scope of the present invention include antibodies, synthetic or native sequence
10 peptides and small molecule antagonists which bind to the B cell marker, optionally
conjugated with or fused to a cytotoxic agent.

"B cell depleting antibody" is an antibody that, upon in vivo adrninistration,
reduces the number of circulating B cells. Preferably, depletion will occur within
about 24 hours of administration to levels which are at least 50% depletion or more.
15 Most preferably, a cold anti-CD20 antibody will deplete B cells substantially as

efficiently (within about 80-90% of the level of B cell depletion within same time) as
Rituxan®.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell
mediated reaction in which nonspecific cytotoxic cells that express Fc receptors

20 (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express FcyRTH only, whereas
monocytes express FcyRI, FcyRH and FcyRHI. FcR expression on hematopoietic cells
in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol

25 9:457-92 (1 99 1 ). To assess ADCC activity of a molecule of interest, an in vitro

ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest maybe assessed in vivo, e.g., in a animal model

30 such as that disclosed in Clynes et al. PNAS (USA) 95 :652-656 (1 998).

"Human effector cells" are leukocytes which express one or more FcRs and
perform effector functions. Preferably, the cells express at least FcyRTJI and perform

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ADCC effector function. Examples of human leukocytes which mediate ADCC
include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being
preferred. The effector cells may be isolated from a native source thereof, e.g. from
5 blood or PBMCs as described herein.

The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native sequence human FcR.
Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor)
and includes receptors of the FcyRI, FcyRII, and FcyRII subclasses, including allelic

1 0 variants and alternatively spliced forms of these receptors. FcyRII receptors include
FcyRUA (an "activating receptor") and FcyRUB (an "inhibiting receptor"), which
have similar amino acid sequences that differ primarily in the cytoplasmic domains
thereof. Activating receptor FcyRCA contains an immunoreceptor tyrosine-based
activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRUB

15 contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic
domain, (see review M. in Daeon, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs
are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et
al., hnmunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.
126:330-41 (1995). Other FcRs, including those to be identified in the future, are

20 encompassed by the term "FcR" herein. The term also includes the neonatal receptor,
FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al,
J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

"Complement dependent cytotoxicity" or "CDC" refers to the ability of a
molecule to lyse a target in the presence of complement. The complement activation

25 pathway is initiated by the binding of the first component of the complement system
(Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen. To assess
complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996), maybe performed.

"Growth inhibitory" antagonists are those which prevent or reduce

30 proliferation of a cell expressing an antigen to which the antagonist binds. For

example, the antagonist may prevent or reduce proliferation of B cells in vitro and/or
in vivo.

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Antagonists which "induce apoptosis" are those which induce programmed
cell death, e.g. of a B cell, as determined by binding of annexin V, fragmentation of
DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or
formation of membrane vesicles (called apoptotic bodies).
5 The term "hypervariable region" when used herein refers to the amino acid

residues of an antibody which are responsible for antigen-binding. The hypervariable
region comprises amino acid residues from a "complementarity determining region"
or "CDR" (e.g. residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain

1 0 variable domain; Kabat et al, Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))
and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (LI), 50-52
(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2)
and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk.l. Mol. Biol.

15 196:901-917 (1987)). "Framework" or 'TR" residues are those variable domain
residues other than the hypervariable region residues as herein denned.

An antagonist "which binds" an antigen of interest, e.g. a B cell surface
marker, is one capable of binding that antigen with sufficient affinity such that the
antagonist is useful as a therapeutic agent for targeting a cell, i.e. a B cell, expressing

20 the antigen.

An "anti-CD20 antibody" herein is an antibody that specifically binds CD20
antigen, preferably human CD20, having measurable B cell depleting activity,
preferably having at least about 10%, more preferably at least 50%, and still more
preferably at least 90%, the B cell depleting activity of RITUXAN® (see U.S. Patent

25 No. 5,736,137, incorporated by reference herein in its entirety).

An "anti-CD22 antibody" herein is an antibody that specifically binds CD22
antigen, preferably human CD22, having measurable B cell depleting activity,
preferably having at least about 10% the B cell depleting activity of RITUXAN® (see
U.S. Patent No. 5,736,137, incorporated by reference herein in its entirety).

30 Specific examples of antibodies which bind the CD20 antigen include':

"Rituximab" ("RITUXAN®") (US PatentNo. 5,736,137, expressly incorporated
herein by reference); yttrium-[90]-labeled 2B8 murine antibody "Y2B8" (US Patent

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No. 5,736,137, expressly incorporated herein by reference); murine IgG2a"Bl"
optionally labeled with 131 I labeled Bl antibody (BEXXARTM) (US Patent No.
5,595,721, expressly incorporated herein by reference); murine monoclonal antibody
"1F5" (Press et al. Blood 69(2):584-591 (1987); and "chimeric 2H7" antibody (US
5 Patent No. 5,677, 1 80, expressly incorporated herein by reference).

Specific examples of antibodies which bind CD22 include Lymphocide™
reported by Immunomedics, now in clinical trials for non-Hodgkin's lymphoma.

The terms "rituximab" or "PJTUXAN®" herein refer to the genetically
engineered chimeric murme/human monoclonal antibody directed against the CD20
10 antigen and designated "C2B8" in US Patent No. 5,736,B7, expressly incorporated
herein by reference. The antibody is an IgGI kappa immunoglobulin amtaining
murine light and heavy chain variable region sequences and human constant region
sequences. Rituximab has a binding affinity for the CD20 antigen of approximately
8.0 nM.

15 An "isolated" antagonist is one which has been identified and separated and/or

recovered from a component of its natural environment. Contaminant components of
its natural environment are materials which would interfere with diagnostic or
therapeutic uses for the antagonist, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antagonist

20 will be purified (1) to greater than 95% by eight of antagonist as determined by the
Lowry method, and most preferably more than 99% by weight, (2) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal arnino acid sequence
by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.

25 Isolated antagonist includes the antagonist in situ within recombinant cells since at
least one component of the antagonist's natural environment will not be present.
Ordinarily, however, isolated antagonist will be prepared by at least one purification
step.

"Mammal" for purposes of treatment refers to any animal classified as a
30 mammal, including humans, domestic and farm animals, and zoo, sports, or pet
animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
"Treatment" refers to both therapeutic treatment and prophylactic or

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preventative measures. Those in need of treatment include those already with the
disease or disorder as well as those in which the disease or disorder is to be prevented.
Hence, the mammal may have been diagnosed as having the disease or disorder or
maybe predisposed or susceptible to the disease.
5 B Cell Malignancy

According to the present invention this includes any B cell malignancy, e.g., B
cell lymphomas and leukemias. Preferred examples include Hodgkin's disease (all
forms, e.g., relapsed Hodgkin's disease, resistant Hodgkin's disease) non-Hodgkin's
lymphomas (low grade, intermediate grade, high grade, and other types). Examples

10 include small lymphocytic/B cell chronic lymphocytic leukemia (SLL/B-CLL),
lymhoplasmacytoid lymphoma (LPL), mantle cell lymphoma (MCL), follicular
lymphoma (FL), diffuse large cell lymphoma (DLCL), Burkitt's lymphoma (BL),
AIDS- related lymphomas, monocytic B cell lymphoma, angioimmunoblastic
lymphoadenopathy, small lymphocytic, follicular, diffuse large cell, diffuse small

15 cleaved cell, large cell immunoblastic lymphoblastoma, small, non-cleaved, Burkitt's
and non-Burkitt's, follicular, predominantly large cell; follicular, predominantly small
cleaved cell; and follicular, mixed small cleaved and large cell lymphomas. See,
Gaidono et al., "Lymphomas", IN CANCER: PRINCIPLES & PRACTICE OF
1 ONCOLOGY, Vol. 2: 213 1-2145 (DeVita et al., eds., 5 th ed. 1997).

20 Other types of lymphoma classifications include immunocytomal

Waldenstrom's MALT-type/monocytoid B cell, mantle cell lymphoma B-CLL/SLL,
diffuse large B-cell lymphoma, follicular lymphoma, and precursor B-LBL.

As noted, B cell malignancies further include especially leukemias such as
ALI^L3 (Burkitt's type leukemia), chronic lymphocytic leukemia (CLL), chronic

25 leukocytic leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, lymphoblastic
leukemia, lymphocytic leukemia, monocytic leukemia, myelogenous leukemia, and
promyelocytic leukemia and monocytic cell leukemias.

"Autoimmune disease" herein includes any autoimmune disease wherein

30 elimination or depletion or inhibition of the activity or proliferation of B cells is

therapeutically beneficial. Such autoimmune diseases will include in particular T and
B cell mediated autoimmune diseases. Examples thereof include: the treatment or

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prevention of autoimmune, inflammatory, proliferative and hyperproliferative
diseases, and of cutaneous manifestations of immunologically medicated diseases
(e.g., rheumatoid arthritis, lupus erythematosus, systemic lupus erythematosus,
Hashimotos thyroiditis, multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis,
5 nephrotic syndrome, psoriasis, atopical dermatitis, contact dermatitis and further
eczematous dermatitides, seborrheic dermatitis, Lichen planus, Pemphigus, bullous
pemphigus, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythema,
cutaneous eosinophilias, Alopecia areata, etc.); the treatment of reversible obstructive
airways disease, intestinal inflammations and allergies (e.g., inflammatory bile

10 disease, Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis, Crohn's
disease and ulcerative colitis), food-related allergies (e.g., migraine, rhinitis and
eczema), and other types of allergies.

Also, the subject combination therapy is useful for treating malignancies,
particularly solid tumors or late stage malignancies wherein B cells promote tumor

1 5 growth, maintenance and/or metastasis but wherein B cells are not themselves the
origin of the malignancy (not B cell malignancy such as non-Hodgkin's lymphoma).

Cell therapy includes any therapy wherein a potentially immunogenic cell is
introduced into a subject, e.g. isogeneic, allogeneic or xenogeneic, which potentially
may contain a tetrologus gene, e.g. one encoding a therapeutic polypeptide.

20 Gene therapy includes any therapy wherein a DNA or RNA sequence is

introduced that modulates (inhibits or enhances) or provides for the expression of a
gene normally or not normallyl expressed, e.g. one involved in a disease. Typically,
the DNA or RNA will be comprised in a vector, e.g. plasmid, virus or in the genome
of a cell, e.g. mammalian cell. Alternatively, the DNA or RNA may be "naked" or

25 comprised in a stabilizing or targeting material, e.g. liposome. Examples include
adenoviral, poxviruses, and other viral vectors, liposomal DNA formulations, etc.

The expression "therapeutically effective amount" refers to an amount of the
naked antibody or radiolabeled antibody which is effective for preventing,
ameliorating or treating the disease in question, e.g. B cell malignancy.

30 The term "immunosuppressive agent" as used herein for adjunct therapy refers

to substances that act to suppress or mask the immune system of the mammal being
treated herein. This would include substances that suppress cytokine production,

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downregulate or suppress self-antigen expression, or mask the MHC antigens.
Examples of such agents include 2-ammo-6-aryl-5-substimtedpyrirrudines (see U.S.
Pat. No. 4,665,077, the disclosure of which is incorporated herein by reference),
azatbioprine; cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde
5 (which masks the MHC antigens, as described in U.S. Pat. No. 4, 120,649);

anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A;
steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, and
dexamethasone; cytokine or cytokine receptor antagonists including anti-interferon-a,
P- or 8-antibodies, anti-tumor necrosis factor-a antibodies, anti-tumor necrosis

10 factor-P antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;
anti-LFA-1 antibodies, including anti-CDl la and anti-CD 18 antibodies; anti-L3T4
antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably
anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding
domain (WO 90/08187 published 7/26/90), streptolanase; TGF-P; streptodornase;

15 RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell
receptor (Cohen et al, U.S. Pat. No. 5,1 14,721); T-cell receptor fragments (Offher et
al, Science, 251: 430-432 (1991); WO 90/11294; Laneway, Nature, 341: 482 (1989);
and WO 91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or

20 prevents the function of cells and/or causes destruction of cells. The term is intended
to include radioactive isotopes (e.g. 123 1, 125 1, 131 I, lu Tn, 131 Tn, 52 P, "C, 67 Cu, 2u At,
,77 Lu, "Y, ,86 Re, 212 Pb, 212 Bi, 47 Sc, 105 Rh, 104 Pd, 153 Sm, 188 Re, 199 Au, 2n At and 213 Bi),
chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically
active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

25 A "chemotherapeutic agent" is a chemical compound useful in the treatment of

cancer. Examples of chemotherapeutic agents include alkylating agents such as
thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and memylamelamines mcluding dfretarnine,

30 triemylenemelamine, trietylenephosphoramide, rriethylenethiophosphaorarnide and
trimethylolomelarnime nitrogen mustards such as chlorambucil, chlomaphazine,
cholophosphamide, estramustine, ifosfamide, mecUoremamine, mecWoremamine

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oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,
5 carabicin, carminomycin, carzinophilin, chromomycins, dactmomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;

10 anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid

analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs
such as fludarabine, 6-mercaptopurine, thiamiprine, unoguanine; pyrirmdme analogs
such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyiuidine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone,

15 dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as ammoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic
acid; aceglatone; aldophosphamide glycoside; arninolevulinic acid; amsacrine;
bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfoiruthine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; Ientinan;

20 lomdamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;

phenamet; pirarubicin; podophyllinic acid'; 2-ethylhydrazide; procarbazine; PSK®;
razoxane; sizofiran; spirogermanium; tenua?onic acid; triaziquone; 2,
2^2"-tricUorotriemylamine; urethan; vindesine; dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");

25 cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb
Oncology, Princeton, NJ) and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony,
France); chlorambucil; gemcitabine; 6-tmoguanine; mercaptopurine; methotrexate;
platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide
(VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;

30 novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT1 1;
topoisomerase inhibitor RFS 2000; d^fluoromemylormthine (DMFO); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives

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of any of the above. Also included in this definition are anti-hormonal agents that act
to regulate or inhibit hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,
4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene
5 (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprohde,
and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the
above.

The term "cytokine" is a generic term for proteins released by one cell
population which act on another cell as intercellular mediators. Examples of such

10 cytokines are lymphokines, monokines, and traditional polypeptide hormones.

Included among the cytokines are growth hormone such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone; parathyroid
hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones
such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and

15 luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin;
placental lactogen; tumor necrosis factor-oc and -p; mullerian-inhibiting substance;
mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth
factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-13;
platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and

20 TGF-P; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive

factors; interferons such as interferon-a, -p, and -y; colony stimulating factors (CSFs)
such as macrophage-CSF (M-CSF); granulocytemacrophage-CSF (GM-CSF); and
granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-g, EL-4,
IL-5, EL-6, lL-7, IL-8, IL-9, IL-1 1, IL-12, IL-15; a tumor necrosis factor such as

25 TNF-ct or TNF-p; and other polypeptide factors including LIF and kit ligand (KL). As
used herein, the term cytokine includes proteins from natural sources or from
recombinant cell culture and biologically active equivalents of the native sequence
cytokines.

The term "prodrug" as used in this application refers to a precursor or
30 derivative form of a pharmaceutically active substance that is less cytotoxic to tumor
cells compared to the parent drug and is capable of being enzymatically activated or
converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer

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Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting
Belfast (1986) and Stella et al, "Prodrugs: A Chemical Approach to Targeted Drug
Delivery," Directed Drug Delivery, Borchardt et al, (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited to,
5 phosphate-containing prodrugs, ttaophosphate-containing prodrugs, sulfate-containing
prodrugs, pepn^e-containing prodrugs, D-amino acid-modified prodrugs, glycosylated
prodrugs, 1 3-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5fluorouridine

10 prodrugs which can be converted into the more active cytotoxic free drug. Examples
of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention
include, but are not limited to, those chemotherapeutic agents described above.

A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant which is useful for delivery of a drug (such as the

15 antagonists disclosed herein and, optionally, a chemotherapeutic agent) to a mammal.
The components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement of biological membranes.

The term "package insert" is used to refer to instructions customarily included
in commercial packages of therapeutic products, that contain information about the

20 indications, usage, dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
Production of Antibodies

The subject invention uses antibodies to CD20 and CD22. These antibodies
will be provided by known methods. As noted, antibodies to both these antigens are

25 well known.

Exemplary techniques for the production of the antibodies used in accordance
with the present invention are described.
(i) Polyclonal antibodies

Polyclonal antibodies are preferably raised in animals by multiple
30 subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful to conjugate the relevant antigen to a protein that is
immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum

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albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a Afunctional or
derivatizing agent, for example, maleirnidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinhnide (through lysine
residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N=C=NR, where R and R 1
5 are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, or
derivatives by combining, e.g. 100 ug or 5 ug of the protein or conjugate (for rabbits
or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the animals are boosted with

10 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant
by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled
and the serum is assayed for antibody titer. Animals are boosted until the titer
plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but
conjugated to a different protein and/or through a different cross-linking reagent.

1 5 Conjugates also can be made in recombinant cell culture as protein fusions. Also,
aggregating agents such as alum are suitably used to enhance the immune response.
(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising the population are

20 identical except for possible naturally occurring mutations that may be present in
minor amounts. Thus, the modifier "monoclonal" indicates the character of the
antibody as not being a mixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridoma
method first described by Kohler et al, Nature, 256:495 (1975), or may be made by

25 recombinant DNA methods (U.S. Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a
hamster, is immunized as hereinabove described to elicit lymphocytes that produce or
are capable of producing antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes

30 then are fused with myeloma cells using a suitable fusing agent, such as polyethylene
glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp.59-1 03 (Academic Press, 1986)).

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The hybridoma cells thus prepared are seeded and grown in a suitable culture
medium that preferably contains one or more substances that inhibit the growth or
survival of the unfused, parental myeloma cells. For example, if the parental myeloma
cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
5 HPRT), the culture medium for the hybridomas typically will include hypoxanthine,
arninopterin, and mymidine (HAT medium), which substances prevent the growth of
HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable
high-level production of antibody by the selected antibody-producing cells, and are

10 sensitive to a medium such as HAT medium. Among these, preferred myeloma cell
lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1
mouse tumors available from the Salk Institute Cell Distribution Center, San Diego,
CaUfornia USA, and SP-2 or X63-Ag8-653 cells available from the American Type
Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human

15 heteromyeloma cell lines also have been described for the production of human
monoclonal antibodies (Kozbor, J. Immunol., 133:300 1 (1984); Brodeur etal,
Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for

20 production of monoclonal antibodies directed against the antigen. Preferably, the
binding specificity of monoclonal antibodies produced by hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
The binding affinity of the monoclonal antibody can, for example, be

25 determined by the 30 Scatchard analysis of Munson et al, Anal Biochem., 107:220
(1980).

After hybridoma cells are identified that produce antibodies of the desired
specificity, affinity, and/or activity, the clones maybe subcloned by limiting dilution
procedures and grown by standard methods (Goding, Monoclonal Antibodies:
30 Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media
for this purpose include, for example, D-MEM or RPML-1640 medium. In addition,
the hybridoma cells may be grown in vivo as ascites tumors in an animal.

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The monoclonal antibodies secreted by the subclones are suitably separated
from the culture medium, ascites fluid, or serum by conventional immunoglobulin
purification procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity chromatography.
5 DNA encoding the monoclonal antibodies is readily isolated and sequenced

using conventional procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are then transfected

10 into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)
cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the antibody include
Skerra et al, Curr. Opinion in Immunol, 5:256-262 (1993) and Pluckthun, Immunol.

15 Revs., 130:151-188 (1992).

Another method of generating specific antibodies, or antibody fragments,
reactive against a CD20 or CD22 is to screen expression libraries encoding
immunoglobulin genes, or portions thereof, expressed in bacteria with a CD20 or
CD22 protein or peptide. For example, complete Fab fragments, Vh regions and Fv

20 regions can be expressed in bacteria using phage expression libraries. See for

example, Ward et al, Nature 341: 544-546 (1989); Huse et al, Science 246: 1275-
1281 (1989); and McCafferty et al, Nature 348: 552-554 (1990). Screening such
libraries with, for example, a CD22 or CD20 peptide, can identify irnmunoglobulin
fragments reactive with CD22 or CD20. Alternatively, the SCID-hu mouse (available

25 from Genpharm) can be used to produce antibodies or fragments thereof.

In a further embodiment, antibodies or antibody fragments can be isolated
from antibody phage libraries generated using the techniques described in McCafferty
et al, Nature, 348:552-554(1990). Clackson et al, Nature, 352:624-628 (1991) and
Marks et al, J. Mol. Biol, 222:581-597 (1991) describe the isolation of murine and

30 human antibodies, respectively, using phage libraries. Subsequent publications
describe the production of high affinity (nM range) human antibodies by chain
shuffling (Marks et al, Bio/Technology, 10:779-783 (1992)), as well as combinatorial

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infection and in vivo recombination as a strategy for constructing very large phage
libraries (Waterhouse et al, Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these
techniques are viable alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
5 The DNA also may be modified, for example, by substituting the coding

sequence for human heavy- and tight-chain constant domains in place of the
homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, et al, Proc.
Natl Acad. ScL USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for a non-immunoglobulin

10 polypeptide.

Typically, such non-immunoglobulin polypeptides are substituted for the
constant domains of an antibody, or they are substituted for the variable domains of
one antigencombining site of an antibody to create a chimeric bivalent antibody
comprising one antigen-combining site having specificity for an antigen and another

1 5 antigen-combining site having specificity for a different antigen.
(Hi) Humanized antibodies

Methods for humanizing non-human antibodies have been described in the art.
Preferably, a humanized antibody has one or more amino acid residues introduced into
it from a source which is non-human. These non-human amino acid residues are often

20 referred to as "import" residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the method of Winter
and co-workers (Jones et al, Nature, 321:522-525 (1986); Reichmann et al,
Nature,332:323-321 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding sequences of a

25 human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable
domain has been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human antibodies in which
some hypervariable region residues and possibly some FR residues are substituted by

30 residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in
making the humanized antibodies is very important to reduce antigenicity. According

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to the so called "best-fit" method, the sequence of the variable domain of a rodent
antibody is screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the rodent is then accepted
as the human framework region (FR) for the humanized antibody (Suns et al, J.
5 Immunol, 151:2296 (1993); Chotbia et al, J. Mol Biol, 196:901 (1987)). Another
method uses a particular framework region derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains. The same
framework may be used for several different humanized antibodies (Carter et al,
Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Prestae/a/., J. Immunol, 151:2623
10 (1993)).

It is further important that antibodies be humanized with retention of high
affinity for the antigen and other favorable biological properties. To achieve this goal,
according to a preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized products using

1 5 three-dimensional models of the parental and humanized sequences. Three-
dimensional immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits analysis of the likely

20 role of the residues in the functioning of the candidate immunoglobulin sequence, i.
e., the analysis of residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR residues can be selected and combined from the
recipient and import sequences so that the desired antibody characteristic, such as
increased affinity for the target antigen(s), is achieved. In general, the hypervariable

25 region residues are directly and most substantially involved in influencing antigen
binding.

(iv) Ptimatized Antibodies

Another highly efficient means for generating recombinant antibodies is
disclosed by Newman, Biotechnology, 10: 1455-1460(1992). More particularly, this
30 technique results in the generation of primatized antibodies which contain monkey
variable domains and human constant sequences. This reference is incorporated by
reference in its entirety herein. Moreover, this technique is also described in

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commonly assigned U.S. Application No. 08/379,072, filed on January 25, 1995,
which is a continuation of U.S. Serial No. 07/912,292, filed July 10, 1992, which is a
continuation-in-part of U.S. Serial No. 07/856,281, filed March 23, 1992, which is
finally a continuation-in-part of U.S. Serial No. 07/735,064, filed July 25, 1991.
5 08/379,072 and the parent application thereof all of which are incorporated by
reference in their entirety herein.

This technique modifies antibodies such that they are not antigenically rejected
upon administration in humans. This technique relies on immunization of
cynomolgus monkeys with human antigens or receptors. This technique was
10 developed to create high affinity monoclonal antibodies directed to human cell surface
antigens.

Identification of macaque antibodies to human CD20 or CD22 by screening of
phage display libraries or monkey heterohybridomas obtained using B lymphocytes
from CD20 or CD22 immunized monkeys can be performed using the methods

15 described in commonly assigned U.S. Application No. 08/487,550, filed June 7, 1995,
incorporated by reference in its entirety herein.

Antibodies generated using the methods described in these applications have
previously been reported to display human effector function, have reduced
immunogenicity, and long serum half-life. The technology relies on the fact that

20 despite the fact that cynomolgus monkeys are phylogenetically similar to humans,
they still recognize many human proteins as foreign and therefore mount an immune
response. Moreover, because the cynomolgus monkeys are phylogenetically close to
humans, the antibodies generated in these monkeys have been discovered to have a
high degree of amino acid homology to those produced in humans. Indeed, after

25 sequencing macaque immunoglobulin light and heavy chain variable region genes, it
was found that the sequence of each gene family was 85-98% homologous to its
human counterpart (Newman et al., 1992). The first antibody generated in this way,
an anti-CD4 antibody, was 91-92% homologous to the consensus sequence of human
immunoglobulin framework regions (Newman et al. , 1 992).

30 (v) Human antibodies

As an alternative to humanization, human antibodies can be generated. For
example, it is now possible to produce transgenic animals (e.g., mice) that are

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capable, upon immunization, of producing a full repertoire of human antibodies in the
absence of endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain joining region
PH) gene in chimeric and germ-line mutant mice results in complete inhibition of
5 endogenous antibody production. Transfer of the human germ-line immunoglobulin
gene array in such germ line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Mad. Acad. Sci.
USA, 90:255 1 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann
etal, Year in immuno., 7:33 (1993); and US Patent Nos. 5,591,669, 5,589,369 and
10 5,545,807.

Alternatively, phage display technology (McCafferty et al, Nature
348:552-553 (1990)) can be used to produce human antibodies and antibody
fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain genes are

1 5 cloned in-frame into either a major or minor coat protein gene of a filamentous

bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on
the surface of the phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on the functional
properties of the antibody also result in selection of the gene encoding the antibody

20 exhibiting those properties. Thus, the phage mimics some of the properties of the B
cell. Phage display can be performed in a variety of formats; for their review see, e.g.,
Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology
3:564-57 1 (1993). Several sources of V-gene segments can be used for phage display.
Clackson et al, Nature, 352:624-628 (1991) isolated a diverse array of

25 anti-oxazolone antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes from
unimmunized human donors can be constructed and antibodies to a diverse array of
antigens (including self antigens) can be isolated essentially following the techniques
described by Marks et al, JMol Biol, 222:581-597 (1991), or Griffith et al, EMBO

30 J. 12:725-734 (1993). See, also, US Patent Nos. 5,565,332 and 5,573,905.

Human antibodies may also be generated by in vitro activated B cells (see US
Patents 20 5,567,610 and 5,229,275). A preferred means of generating human

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antibodies using SCID mice is disclosed in commonly-owned, co-pending
applications.

(vi) Antibody fragments

Various techniques have been developed for the production of antibody
5 fragments. Traditionally, these fragments were derived via proteolytic digestion of
intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical
Methods 24:107-1 17 (1992) and Brennan et al, Science, 229:81 (1985)). However,
these fragments can now be produced directly by recombinant host cells. For example,
the antibody fragments can be isolated from the antibody phage libraries discussed

10 above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab')2 fragments (Carter et al, Bio/Technology 10:
163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated
directly from recombinant host cell culture. Other techniques for the production of
antibody fragments will be apparent to the skilled practitioner. In other embodiments,

15 the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US
Patent No. 5,571,894; and US Patent No. 5,587,458. The antibody fragment may also
be a "linear antibody", e.g., as described in US Patent 5,641,870 for example. Such
linear antibody fragments maybe monospecific or bispecific.

(vii) Bispecific antibodies

20 Bispecific antibodies are antibodies that have binding specificities for at least

two different epitopes. Exemplary bispecific antibodies may bind to two different
epitopes of the B cell surface marker. Other such antibodies may bind a first B cell
marker and further bind a second B cell surface marker. Alternatively, an anti-B cell
marker binding arm may be combined with an arm which binds to a triggering

25 molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc
receptors for IgG (FcyR), such as FcyRI (CD64), FcyRH (CD32) and FcyRHI (CD 1 6)
so as to focus cellular defense mechanisms to the B cell. Bispecific antibodies may
also be used to localize cytotoxic agents to the B cell. These antibodies possess a B
cell marker-binding arm and an arm which binds the cytotoxic agent (e.g. saporin,

30 anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab)2 bispecific antibodies).

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Methods for making bispecific antibodies are known in the art. Traditional
production of full length bispecific antibodies is based on the coexpression of two
immunoglobulin heavy chain-light chain pairs, where the two chains have different
specificities (Millstein et al, Nature, 305:537-539 (1983)). Because of the random
5 assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules, of which only one has
the correct bispecific structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and the product yields
are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al,

10 EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired
binding specificities (antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an immunoglobulin heavy
chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It

15 is preferred to have the first heavy-chain constant region (CHI) containing the site
necessary for light chain binding, present in at least one of the fusions. DNAs
encoding the immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression vectors, and are
co-transfected into a suitable host organism. This provides for great flexibility in

20 adjusting the mutual proportions of the three polypeptide fragments in embodiments
when unequal ratios of the three polypeptide chains used in the construction provide
the optimum yields. It is, however, possible to insert the coding sequences for two or
all three polypeptide chains in one expression vector when the expression of at least
two polypeptide chains in equal ratios results in high yields or when the ratios are of

25 no particular significance.

In a preferred embodiment of this approach, the bispecific antibodies are
composed of a hybrid immunoglobulin heavy chain with a first binding specificity in
one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a
second binding specificity) in the other arm. It was found that this asymmetric

30 structure facilitates the separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations, as the presence of an immunoglobulin light
chain in only one half of the bispecific molecule provides for a facile way of

WO 01/97858 PCT/US01/18939

separation. This approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et ai, Methods in
Enzymology, 121:210(1986).

According to another approach described in US Patent No. 5,731,168, the
5 interface between a pair of antibody molecules can be engineered to maximize the
percentage of heterodimers which are recovered from recombinant cell culture. The
preferred interface comprises at least a part of the CH3 domain of an antibody
constant domain. In this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger side chains (e.g.
1 0 tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the
large side chains) are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the heterodimer over other
unwanted end-products such as homodimers.

15 Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For

* . f

example, one of the antibodies in the heteroconjugate can be coupled to avidin, the
other to biotin. Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HTV
infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate

20 antibodies may be made using any convenient cross-linking methods. Suitable

cross-linking agents are well known in the art, and are disclosed in US Patent No.
4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have
also been described in the literature. For example, bispecific antibodies can be

25 prepared using chemical linkage. Brennan et al, Science, 229:81(1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2
fragments. These fragments are reduced in the presence of the dithiol complexing
agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to thionitrobenzoate

30 (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the
Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar
amount of the other Fab'-TNB derivative to form the bispecific antibody. The

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bispecific antibodies produced can be used as agents for the selective immobilization
of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragments from
E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al,
5 J.Exp. Med., 175:2 1 7-225 (1 992) describe the production of a fully humanized
bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately secreted
from E. coli and subjected to directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to cells
overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the

1 0 lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments
directly from recombinant cell culture have also been described. For example,
bispecific antibodies have been produced using leucine zippers. Kostelny et al, J.
Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun

1 5 proteins were linked to the Fab' portions of two different antibodies by gene fusion.
The antibody homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. This method can also be utilized
for the production of antibody homodimers. The "diabody" technology described by
Hollinger et al, Proc.Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an

20 alternative mechanism for making bispecific antibody fragments. The fragments
comprise a heavy-chain variable domain (Vh) connected to a light-chain variable
domain (Vijby a linker which is too short to allow pairing between the two domains
on the same chain. Accordingly, the V H and Vl domains of one fragment are forced to
pair with the complementary Vl and Vh domains of another fragment, thereby

25 forming two antigen-binding sites. Another strategy for making bispecific antibody
fragments by the use of single-chain Fv (sFv) dimers has also been reported. See
Gruber etal, J. Immunol, 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared. Tutt et al. J. Immunol 147: 60(1991).

30 Antibody Conjugates and Other Modifications

The subject therapies may first include the administration of antibody other
than the radiolabeled CD22 antibody wherein the antibody is attached, e.g. to a

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cytotoxin or therapeutic moiety conjugate.

Chemotherapeutic agents useful in the generation of such antibody-cytotoxic
agent conjugates have been described above.

Conjugates of an antibody and one or more small molecule toxins, such as a
5 calicheamicin, a maytansine (US Patent No. 5,208,020), a trichothene, and CC 1065
are also contemplated herein. In one preferred embodiment of the invention, the
antagonist is conjugated to one or more maytansine molecules (e.g. about 1 to about
1 0 maytansine molecules per antagonist molecule). Maytansine may, for example, be
converted to May SS-Me which may be reduced to May-SH3 and reacted with

10 modified antagonist (Charm et al. Cancer Research 52:127-131(1992)) to generate a
maytansinoid-antagonist conjugate.

Alternatively, the antibody may be conjugated to one or more caUcheamicin
molecules. The caUcheamicin family of antibiotics are capable of producing double
stranded DNA breaks at sub-picomolar concentrations. Structural analogues of

1 5 calicheamicin which may be used include, but are not limited to, y\, eta, aj,

N-acetyl-yi 1 , PSAG and 0\ (Hinman et al. Cancer Research 53:3336-3342 (1993) and
Lode et al, Cancer Research 58: 2925-2928 (1998)).

Enzymatically active toxins and fragments thereof which can be used include
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain

20 (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,
enomycin and the tricothecenes. See, for example, WO 93/21232 published October

25 28,1993.

The present invention further contemplates antibody conjugated with a
compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such
as a deoxyribonuclease; DNase).

As discussed above, a variety of radioactive isotopes are available for the
30 production of radioconjugated antagonists. Examples include At 211 , 1 131 , 1 125 , Y 90 ,
Re 186 , RE 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using a variety of

WO 01/97858 PCT7US01/18939

bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyriyldithiol)
propionate (SPDP), succinimidyl-4-(N-malehnidomethyl) cyclohexane-l-carboxylate,
iminotbiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipunidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as
5 glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds
(such as 1 ,5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin can be
prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1

10 isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic.acid (MX-DTPA) is an
exemplary chelating agent for conjugation of radionucleotide to the antagonist. See
W094/1 1026. The linker may be a "cleavable linker" facilitating release of the
cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Charm et al. Cancer Research

15 52:127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the antibody and cytotoxic agent
may be made, e.g. by recombinant techniques or peptide synthesis.

In yet another embodiment, the antibody may be conjugated to a "receptor"
(such streptavidin) for utilization in tumor pretargeting wherein the antagonist-

20 receptor conjugate is administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then administration of a
"ligand" (e.g. avidin) which is.conjugated to a cytotoxic agent (e.g. a radionucleotide).

The antibodies of the present invention may also be conjugated with a prodrug
activating enzyme which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,

25 see W081/01 145) to an active anti-cancer drug. See, for example, WO 88/07378 and
U.S. Patent No. 4,975,278.

The enzyme component of such conjugates includes any enzyme capable of
acting on a prodrug in such a way so as to covert it into its more active, cytotoxic
form.

30 Enzymes that are useful in the method of this invention include, but are not

limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs
into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into

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free drugs; cytosine deaminase useful for converting non-toxic5-fluorocytosine into
the anti-cancer drug, fluorouracil; proteases, such as serratia protease, mermolysin,
subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that axe
useful for converting peptide-containing prodrugs into free drugs;
5 D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid
substituents; carbohydratecleaving enzymes such as 13-galactosidase and
neuraminidase useful for converting glycosylated prodrugs into free drugs;
13-lactamase useful for converting drugs derivatized with 13-lactams into free drugs;
and penicillin amidases, such as penicillin V amidase or peniciUin G amidase, useful

1 0 for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or
phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used to convert the
prodrugs of the invention into free active drugs (see, e.g., Massey, Nature
328:457-458 (1987)). Antagonist-abzyme conjugates can be prepared as described

1 5 herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antagonist by
techniques well known in the art such as the use of the heterobifunctional crosslinking
reagents discussed above. Alternatively, fusion proteins comprising at least the
antigen binding region of an antagonist of the invention linked to at least a

20 functionally active portion of an enzyme of the invention can be constructed using
recombinant DNA techniques well known in the art (see, e.g., Neuberger et al,
Nature, 312:604-608 (1984)).

Other modifications of the antibody are contemplated herein. For example, the
antibody may be linked to one of a variety of nonproteinaceous polymers, e.g.,

25 polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol.

The antibodies disclosed herein may also be formulated as liposomes.
Liposomes containing the antagonist are prepared by methods known in the art, such
as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et

30 al, Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and

4,544,545; and W097/38731 published October 23, 1997. Liposomes with enhanced
circulation time are disclosed in U.S. Patent No. 5,013,556.

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Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising phosphatidylcholine,
cholesterol and PEG derivatized phosphatidylethanolamine (PEG-PE). Liposomes are
extruded through filters of defined pore size to yield liposomes with the desired
5 diameter. Fab' fragments of an antibody of the present invention can be conjugated to
the liposomes as described in Martin et al. J. Biol. Chem. 257:286-288 (1982) via a
disulfide interchange reaction. A chemotherapeutic agent is optionally contained
within the liposome. See Gabizon et al. J.National Cancer Inst. 81(19)1484 (1989).
Amino acid sequence modification(s) of protein or peptide antagonists

1 0 described herein are contemplated. For example, it may be desirable to improve the
binding affinity and/or other biological properties of the antibody. Amino acid
sequence variants of the antibody are prepared by introducing appropriate nucleotide
changes into the antibody encoding nucleic acid, or by peptide synthesis. Such
modifications include, for example, deletions from, and/or insertions into and/or

15 substitutions of, residues within the amino acid sequences of the antagonist. Any
combination of deletion, insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of the antagonist, such
as changing the number or position of glycosylation sites.

20 A useful method for identification of certain residues or regions of the

antibody that are preferred locations for mutagenesis is called "alanine scanning
mutagenesis" as described by Cunningham and Wells Science, 244:1081-1085 (1989).
Here, a residue or group of target residues are identified (e.g., charged residues such
as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino

25 acid (most preferably alanine or polyalanine) to affect the interaction of the amino
acids with antigen. Those amino acid locations demonstrating functional sensitivity to
the substitutions then are refined by introducing further or other variants at, or for, the
sites of substitution. Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need not be

30 predetermined. For example, to analyze the performance of a mutation at a given site,
ala scanning or random mutagenesis is conducted at the target codon or region and the
expressed antagonist variants are screened for the desired activity.

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Amino acid sequence insertions include amino- and/or carboxyl-tenninal
fusions ranging in length from one residue to polypeptides containing a hundred or
more residues, as well as intrasequence insertions of single or multiple amino acid
residues. Examples of terminal insertions include an antagonist with an N-terminal
5 methionyl residue or the antagonist fused to a cytotoxic polypeptide. Other insertional
variants of the antagonist molecule include the fusion to the N- or C-terminus of the
antagonist of an enzyme, or a polypeptide which increases the serum half-life of the
antagonist.

Another type of variant is an amino acid substitution variant. These variants
10 have at least one amino acid residue in the antagonist molecule replaced by different
residue. The sites of greatest interest for substitutional mutagenesis of antibody
antagonists include the hypervariable regions, but FR alterations are also
contemplated. Conservative substitutions are shown in Table 1 under the heading of
"preferred substitutions". If such substitutions result in a change in biological activity,
15 then more substantial changes, denominated "exemplary substitutions" in Table 1, or
as further described below in reference to amino acid classes, may be introduced and
the products screened.

WO 01/97858 PCT/US01/18939

Table 1

Original Residue

Exemplary Substitutions

Preferred Substitutions

Ala (A)

val; leu; ile

val

Arg(R)

lys; gin; asn

lys

Asn(N) .

gin; his; asp, lys; arg

gin

Asp(D)

glu; asn

glu

Cys(C)

ser; ala

ser

Gln(Q

asn; glu

asn

Glu(E)

asp; gin

asp

Gly(G)

ala

ffis(H)

asn; gin; lys; arg

arg

Ile (I)

Leu; val; met; ala;
phe; norleucine

leu

Leu(L)

tiorleucine; ile; val;
met; ala; phe

ile

Lys(K)

arg; gin; asn

arg

Met(M)

leu; phe; ile

Leu

Phe(F)

leu; val; ile; ala; tyr

tyr

Pro(P)

ala

Ser(S)

thr

Thr(T)

ser

Trp(W)

tyr; phe

tyr

ryr(Y)

trp; phe; thr; ser 1

phe

Val(V)

ile; leu; met; phe;
ala; norleucine

leu

Substantial modifications in the biological properties of the antibody are
accomplished by selecting substitutions that differ significantly in their effect on
5 maintaining (a) the structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
Naturally occurring residues are divided into groups based on common side-chain
properties:

10 (1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophiuic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and
15 (6) aromatic: trp, tyr, phe.

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Non-conservative substitutions will entail exchanging a member of one of
these classes for another class.

Any cysteine residue not involved in maintaining the proper conformation of
the antagonist also may be substituted, generally with serine, to improve the oxidative
5 stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine
bonds) may be added to the antagonist to improve its stability (particularly where the
antagonist is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a humanized or

10 human antibody). Generally, the resulting variants selected for further development
will have improved biological properties relative to the parent antibody from which
they are generated. A convenient way for generating such substitutional variants is
affinity maturation using phage display. Briefly, several hypervariable region sites
(e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site.

15 The antibody variants thus generated are displayed in a monovalent fashion from

filamentous phage particles as fusions to the gene HI product of Ml 3 packaged within
each particle. The phage-displayed variants are then screened for their biological
activity (e.g. binding affinity) as herein disclosed. In order to identify candidate
hypervariable region sites for modification, alanine scanning mutagenesis can be

20 performed to identified hypervariable region residues contributing significantly to

antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal
structure of the antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring residues are candidates
for substitution according to the techniques elaborated herein. Once such variants are

25 generated, the panel of variants is subjected to screening as described herein and

antibodies with superior properties in one or more relevant assays may be selected for
further development.

Another type of amino acid variant of the antibody alters the original
glycosylation pattern of the antagonist. By altering is meant deleting one or more

30 carbohydrate moieties found in the antagonist, and/or adding one or more
glycosylation sites that are not present in the antagonist.

Glycosylation of polypeptides is typically either N-linked or O-linked.

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N-linked refers to the attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-
X-threonine, where X is any amino acid except proline, are the recognition sequences
for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
5 Thus, the presence of either of these tripeptide sequences in a polypeptide creates a
potential glycosylation site. O-linked glycosylation refers to the attachment of one of
the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most
commonly seine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.

1 0 Addition of glycosylation sites to the antibody is conveniently accomplished

by altering the amino acid sequence such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation sites). The alteration
may also be made by the addition of, or substitution by, one or more seine or
threonine residues to the sequence of the original antagonist (for O-linked

15 glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of the antibody
are prepared by a variety of methods known in the art. These methods include, but are
not limited to, isolation from a natural source (in the case of naturally occurring amino
acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed)

20 mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the antagonist.

It may be desirable to modify the antibodies used in the invention to improve
effector function, e.g. so as to enhance antigen-dependent cell-mediated cyotoxicity
(ADCC) and/or complement dependent cytotoxicity (CDC) of the antagonist. This

25 may be achieved by introducing one or more amino acid substitutions in an Fc region
of an antibody antagonist. Alternatively or additionally, cysteine residue(s) may be
introduced in the Fc region, thereby allowing interchain disulfide bond formation in
this region. The homodimeric antibody thus generated may have improved
intemaUzation capability and/or increased complement- mediated cell killing and

30 antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med.
176:1 191-1 195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity may also be prepared

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using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc
regions and may thereby have enhanced complement lysis and ADCC capabilities.
See Stevenson et al. Anti-Cancer Drug Design 3:2 19-230 (1989).
5 To increase the serum half life of the antibody, one may incorporate a salvage

receptor binding epitope into the antibody (especially an antibody fragment) as
described in US Patent 5,739,277, for example. As used herein, the term "salvage
receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule
(e.g„ IgGI, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum

1 0 half-life of the IgG molecule.
Pharmaceutical Formulations

Therapeutic formulations comprising cold anti-CD20 and radiolabeled anti.-
CD22 antibodies used in accordance with the present invention are prepared for
storage by mixing an antagonist having the desired degree of purity with optional

15 pharmaceutically acceptable carriers, excipients or stabilizers (Remington 's

Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, and other organic acids; antioxidants including

20 ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl

ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular
weight (less than about 10 residues) polypeptides; proteins, such as serum albumin,

25 gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol,
trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g.

30 Zn-protein complexes); and/or non-ionic surfactants such as T WEEN™,
PLURONICS™ or polyethylene glycol (PEG).

The antibodies may be in the same formulation or may be administered in

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difficult formulations. Administration can be concurrent or sequential, and may be
effective in either order.

Exemplary anti-CD20 antibody formulations are described in W098/56418,
expressly incorporated herein by reference. This publication describes a liquid
5 multidose formulation comprising 40 mg/mL rituximab, 25 mM acetate, 150 mM
trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum
shelf life of two years storage at 2-8°C. Another anti-CD20 formulation of interest
comprises 1 Omg/mL rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium
citrate dihydrate, 0.7mg/mL polysorbate 80, and Sterile Water for Injection, pH 6.5.

10 Lyophilized formulations adapted for subcutaneous administration are

described in W097/04801 Such lyophilized formulations maybe reconstituted with a
suitable diluent to a high protein concentration and the reconstituted formulation may
be ad^ninistered subcutaneously to the mammal to be treated herein.

The formulation herein may also contain more than one active compound as

1 5 necessary for the particular indication being treated, preferably those with

complementary activities that do not adversely affect each other. For example, it may
be desirable to further provide a chemotherapeutic agent, cytokine or
immunosuppressive agent (e.g. one which acts on T cells, such as cyclosporin or an
antibody that binds T cells, e.g. one which binds LFA-1). The effective amount of

20 such other agents depends on the amount of antagonist present in the formulation, the
type of disease or disorder or treatment, and other factors discussed above. These are
generally used in the same dosages and with adnunistration routes as used
hereinbefore or about from 1 to 99% of the heretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared, for

25 example, by 30 coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules)
or in macroemulsions. Such techniques are disclosed in Remington 's Pharmaceutical

30 Sciences 1 6th edition, Osol, A. Ed. (1 980).

Sustained-release preparations may be prepared. Suitable examples of
sustained release preparations include semipermeable matrices of solid hydrophobic

WO 01/97858 PCT/US01/18939

polymers containing the antagonist, which matrices are in the form of shaped articles,
e.g. films, or microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
5 acid and y ethyl-L-glutamate, noir degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate),
and poly-D-(-)-3-hydroxybutyric acid. The formulations to be used for in vivo
adrninistration must be sterile. This is readily accomplished by filtration through

1 0 sterile filtration membranes.

Treatment with Cold Anti-CD20 and Hot (Radiolabeled) Anti-CD22 Antibody or
Antibody Fragment

A composition comprising cold CD20 antibody, e.g. Rituxan® as a
radiolabeled anti-CD22 antibody, preferably 90 Y (radiolabeled by use of MXDTPA as

15 the chelate) will be formulated, dosed, and a<irninistered in a fashion consistent with
good medical practice. Factors for consideration in this context include the particular
B cell malignancy, or other condition, e.g. autoimmune, allergy, inflammatory
disorder, cell therapy or gene therapy, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the disease or disorder, the

20 site of delivery of the agent, the method of administration, the scheduling of

administration, and other factors known to medical practitioners. The therapeutically
effective amount of the antagonist to be administered will be governed by such
considerations.

The CD20 antibody and the radiolabeled CD22 antibody may be in the same
25 or in different formulations. These formulations can be administered separately or
concurrently, and in either order. Preferably, the cold CD20 antibody will be
adtninistered separately from the radiolabeled CD22.

As a general proposition, the therapeutically effective amount of an antibody
administered parenterally per dose will typically be in the range of about 0. 1 to 500
30 mg/kg of patient body weight per day, with the typical initial range of antagonist used
being in the range of about 2 to 1 00 mg/kg.

The preferred anti-CD20 antibody is PJTUXAN®. Suitable dosages for such

WO 01/97858

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antibody are, for example, in the range from about 20mg/m2 to about l'000mg/m2.
The dosage of the antibody may be the same or different from that presently
recommended for RITUXAN® for the treatment of non-Hodgkin's lymphoma. For
example, one may administer to the patient one or more doses of substantially less
5 than 375mg/m2 of the antibody, e.g. where the dose is in the range from about

20mg/m 2 to about 250mg/m 2 , for example from about 50mg/m 2 to about 200mg/m 2 .
The amount of the radiolabeled anti-CD22 antibody will depend upon factors such as
the particular therapeutic radiolabel, e.g. whether it is an a, P or 8 emitter. Methods
for determining appropriate dosages of radiation are well known. Preferably, a dosage

10 will be selected that does not result in myelosuppression severe enough to require one
marrow or stem cell transplant.

Preferably, the anti-CD20 antibody will possess substantially B cell deleting
activity and will induce apoptosis of B cells, comparable to Rituxan®.

Moreover, one may administer one or more initial doses of the CD20 or

15 radiolabeled anti-CD22 antibody followed by one or more subsequent dose(s),

wherein the mg/m 2 dose of the antibody in the subsequent doses) exceeds the mg/m 2
dose of the antibody in the initial dose(s). For example, the initial dose may be in the
range from about 20mg/m 2 to about 250mg/m 2 (e.g. from about 50mg/m 2 to about
200mg/m 2 ) and the subsequent dose maybe in the range from about 250mg/m 2 to

20 about 1000mg/m 2 .

As noted above, however, these suggested amounts of both CD20 and CD22
antibody are subject to a great deal of therapeutic discretion. The key factor in
selecting an appropriate dose and scheduling is the result obtained, as indicated above.
For example, relatively higher doses may be needed initially for the treatment of

25 ongoing and acute diseases. To obtain the most efficacious results, depending on the
particular B cell malignancy, the antagonist is administered as close to the first sign,
diagnosis, appearance, or occurrence of the disease or disorder as possible or during
remissions of the disease or disorder.

The antibodies are administered by any suitable means, including parenteral,

30 subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous

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administration. In addition, the antibody may suitably be administered by pulse
infusion, e.g., with declining doses of the antibody. Preferably the dosing is given by
injections, most preferably intravenous or subcutaneous injections, depending in part
on whether the a(lministration is brief or chronic.
5 One additionally may administer other compounds, such as chemotherapeutic

agents, immunosuppressive agents and/or cytokines with the antibodies herein. The
combined administration includes co-administration, using separate formulations or a
single pharmaceutical formulation, and consecutive administration in either order,
wherein preferably there is a time period while both (or all) active agents

10 simultaneously exert their biological activities.

Aside from administration of antibodies to the patient the present application
contemplates administration of antibodies by gene therapy. Such administration of
nucleic acid encoding the antibodies is encompassed by the expression "admimstering
a therapeutically effective amount of an antagonist". See, for example, W096/07321

15 published March 14, 1996 concerning the use of gene therapy to generate intracellular
antibodies.

There are two major approaches to getting the nucleic acid (optionally
contained in a vector) into the patient's cells; in vivo and ex vivo. For in vivo delivery
the nucleic acid is injected directly into the patient, usually at the site where the

20 antagonist is required. For ex vivo treatment, the patient's cells are removed, the
nucleic acid is introduced into these isolated cells and the modified cells are
administered to the patient either directly or, for example, encapsulated within porous
membranes which are implanted into the patient (see, e.g. U.S. Patent Nos. 4,892,538
and 5,283,187). There are a variety of techniques available for introducing nucleic

25 acids into viable cells. The techniques vary depending upon whether the nucleic acid
is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro
include the use of liposomes, electroporation, microinjection, cell fusion,
DEAF-dextran, the calcium phosphate precipitation method, etc. A commonly used

30 vector for ex vivo delivery of the gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques include
transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno

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associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Choi, for example). In some situations it is
desirable to provide the nucleic acid source with an agent that targets the target cells,
such as an antibody specific for a cell surface membrane protein or the target cell, a
5 ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins
which bind to a cell surface membrane protein associated with endocytosis may be
used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof
tropic for a particular cell type, antibodies for proteins which undergo internalization
in cycling, and proteins that target intracellular localization and enhance intracellular

1 0 half-life. The technique of receptor-mediated endocytosis is described, for example,
by Wu et al, .1. Biol. Chem 262:4429-4432 (1987); and Wagner et al, Proc. Natl.
Acad. Sci. USA 87:3410-3414(1990). For review of the currently known gene
marking and gene therapy protocols see Anderson et al, Science 256:808-8 13 (1992).
See also WO 93/25673 and the references cited therein.

15 Articles of Manufacture

In another embodiment of the invention, an article of manufacture containing
materials useful for the treatment of the diseases or disorders described above is
provided.

The article of manufacture comprises a container and a label or package insert
20 on or associated with the container. Suitable containers include, for example, bottles,
vials, syringes, etc. The containers maybe formed from a variety of materials such as
glass or plastic. The container holds or contains a composition which is effective for
treating the disease or disorder of choice and may have a sterile access port (for
example the container may be an intravenous solution bag or a vial having a stopper
25 pierceable by a hypodermic injection needle). As whole, there may be one or several
compositions. At least one active agent in one of those compositions is a cold CD20
antibody, preferably one having substantial B cell depleting activity and at least one
antibody is therapeutically radiolabeled anti-CD22 antibody or fragment, preferably
90 Y radiolabeled antibody. The label or package insert indicates that the composition
30 is used for treating a patient having or predisposed to B cell malignancy, such as those
listed hereinabove or other conditions or treatment wherein inhibition of B cells is
desirable, e.g. autoimmune disease, transplant, gene therapy, cell therapy or

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inflammatory condition. The article of manufacture may further comprise a second
container comprising a pharmaceutically acceptable buffer, such as bacteriostatic
water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose
solution. It may further include other materials desirable from a commercial and user
5 standpoint, including other buffers, diluents, filters, needles, and syringes.

Further details of the invention are illustrated by the following non-limiting
Examples. The disclosures of all citations in the specification are expressly
incorporated herein by reference.

The antibodies of the invention may be administered to a human or other
10 animal in accordance with the aforementioned methods of treatment in an amount
sufficient to produce such effect to a therapeutic or prophylactic degree. Such
antibodies of the invention can be administered to such human or other animal in a
conventional dosage form prepared by combining the antibody of the invention with a
conventional pharmaceutically acceptable carrier or diluent according to known
1 5 techniques. It will be recognized by one of skill in the art that the form and character
of the pharmaceutically acceptable carrier or diluent is dictated by the amount of
active ingredient with which it is to be combined, the route of administration and
other well-known variables.

The routine of administration of the antibody (or fragment thereof) of the
20 invention may be oral, parenteral, by inhalation or topical. The term parenteral as
used herein includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal
or vaginal administration. The subcutaneous and intramuscular forms of parenteral
administration are generally preferred.

The daily parenteral and oral dosage regimes for employing compounds of the
25 invention to prophylactically or therapeutically induce immunosuppression, or to
therapeutically treat carcinogenic tumors will generally be in the range of about 0.05
to 100, but preferably about 0.5 to 10, milligrams per kilogram body weight per day.

The antibodies of the invention may also be administered by inhalation. By
"inhalation" is meant intranasal and oral inhalation administration. Appropriate
30 dosage forms for such administration, such as an aerosol formulation or a metered
dose inhaler, may be prepared by conventional techniques. The preferred dosage

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amount of a compound of the invention to be employed is generally within the range
of about 10 to 100 milligrams.

The antibodies of the invention may also be administered topically. By topical
administration is meant non-systemic administration and includes the application of
5 an antibody (or fragment thereof) compound of the invention externally to the
epidermis, to the buccal cavity and instillation of such an antibody into the ear, eye
and nose, and where it does not significantly enter the blood stream. By systemic
administration is meant oral, intravenous, intraperitoneal and intramuscular
administration. The amount of an antibody required for therapeutic or prophylactic

10 effect will, of course, vary with the antibody chosen, the nature and severity of the
condition being treated and the animal.
Combination Therapy of Invention

The present invention relates to the combined use of a cold anti-CD20
antibody and a radiolabeled anti-CD22 antibody targeted to extracellular determinants

15 of CD20 and CD22. CD20 and CD22 are both antigens present on B-cells. These
antibodies are selectively reactive under immunological conditions to those
determinants of CD20 and CD22 displayed on the surface of B-cells and accessible to
the antibody from the extracellular milieu.

The term "selectively reactive" or "specific to" includes reference to the

20 preferential association of an antibody, in whole or part, with a cell or tissue bearing
the CD22 or CD20 target molecule and not to cells or tissues lacking that target
molecule. It is, of course, recognized that a certain degree of nonspecific interaction
may occur between a molecule and a non-target cell or tissue. Nevertheless, specific
binding, may be distinguished as mediated through specific recognition of the target

25 CD22 or CD20 molecule. Typically specific binding results in a much stronger
association between the delivered molecule and cells bearing CD22 or CD20 than
between the bound molecule and cells lacking CD22 or CD20. Specific binding
typically results in greater than two-fold, preferably greater than five-fold, more
preferably greater than ten-fold and most preferably greater than one hundred-fold

30 increase in amount of bound ligand (per unit time) to a cell or tissue bearing CD22 or
CD20 as compared to a cell or tissue lacking CD22 or CD20. Specific binding to a
protein under such conditions requires an antibody that is selected for its specificity

WO 01/97858 PCT/US01/18939

for a particular protein. A variety of immunoassay formats are appropriate for
selecting antibodies specifically immunoreactive with a particular protein. For
example, solid-phase ELISA immunoassays are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See Harlow and Lane (1 988)
5 Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used to determine
specific immunoreactivity.
Anti-CD20 Antibodies

The anti-CD20 antibody preferably will comprise a chimeric, humanized or

10 human monoclonal antibody that specifically bind CD20 or a fragment thereof. Most
preferably, the anti-CD20 antibody will comprise RITUXAN®, the nucleic acid
sequence and amino acid sequence of which is reported in U.S. Patent No. 5,736,137,
incorporated by reference in its entirety herein. This chimeric anti-CD20 antibody
very effectively depletes B-cells and has been approved for use by the FDA for

15 treating non-Hodgkin's lymphoma. However, humanized and human monoclonal
antibodies may also be used.

The radiolabeled anti-CD22 antibody will preferably comprise a chimeric,
humanized or human monoclonal antibody or binding fragment thereof specific to
CD22. In the preferred embodiment, a 90 Y radiolabeled humanized monoclonal

20 antibody, the sequence of which is disclosed in Leung et al in U.S. Patent 5,789,554,
issued August 4, 1998, will be utilized. This reference is also incorporated by
reference in its entirely herein. However, other monoclonal antibodies and binding
fragments can be substituted therefor.

Additionally, the use of radionuclides other than 90 Y is contemplated, e.g., 131 I,

25 67 Cu, 32 P, 125 1, 186 Re, 188 Re, 211 At and the like. Suitable radioisotopes include a, p,
and y-emitters, Auger electron emitters, and neutron capturing agents that emit a-
particles or a radioisotype that decays by electron capture.

The radiolabel may be attached directly or indirectly to the antibody or
fragment, e.g., by use of a chelating agent. Suitable chelators include by way of

30 example DTPA and DETA.

WO 01/97858

PCT7US01/18939

Suitable antibody fragments include any antibody which lacks substantially all
the Fc region of a native antibody. These include in particular scFv, dsFv, Fab,
FCab 1 )^ F(ab) 2 , Fab, and the like.

As noted, the anti-CD20 antibody and radiolabeled anti-CD22 antibody can be
5 administered separately or in combination, and in either order. Preferably, the anti-
CD20 antibody is administered initially in therapeutically effective amounts, followed
by the radiolabeled anti-CD22 antibody.
Binding Affinity of Antibodies

The antibodies used in this invention are capable of specifically binding an

1 0 extracellular epitope of CD22 or CD20. An anti-CD22 or anti-CD20 antibody has
binding affinity for CD22 or CD20 if the antibody binds or is capable of binding
CD22 or CD20 as measured or determined by standard antibody-antigen assays, for
example, competitive assays, saturation assays, or standard immunoassays such as
ELISA or RIA. This definition of specificity applies to single heavy and/or light

15 chains, CDRS, fusion proteins or fragments of heavy and/or light chains, that are
specific for CD22 or CD20 if they bind CD22 alone or in combination.

In competition assays the ability of an antibody to bind a ligand is determined
by detecting the ability of the antibody to compete with the binding of a compound
known to bind the ligand. Numerous types of competitive assays are known and are

20 discussed herein. Alternatively, assays that measure binding of a test compound in the
absence of an inhibitor may also be used. For instance, the ability of a molecule or
other compound to bind CD22 can be detected by labeling the molecule of interest
directly or the molecule be unlabeled and detected indirectly using various sandwich
assay formats. Numerous types of binding assays such as competitive binding assays

25 are known (see, e.g., U.S. Patent Nos. 3,376,1 10 and 4,016,043, and Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, N.Y.
(1988), which are incorporated herein by reference). Assays for measuring binding of
a test compound to one component alone rather than using a competition assay are
also available. For instance, antibodies can be used to identify the presence of the

30 ligand. Standard procedures for monoclonal antibody assays, such as ELISA, may be
used (see, Harlow and Lane, supra). For a review of various signal producing systems

WO 01/97858

PCT/US01/18939

which may be used see, U. Patent No. 4,391,904, which is incorporated herein by
reference.

The dosages of anti-CD20 to be used in the present invention may vary
depending on the patient and the antibody used. Chimeric anti-CD20 antibody such
5 as RITUXIMAB® may be administered at a dosage of at least about 50 mg/m 2 weekly
for at least 4 weeks. A particularly preferred dosage regimen is about 375 mg/m 2
weekly for four weeks.

As noted, preferably after administration of the anti-CD20 antibody, a
radiolabeled anti-CD22 antibody will be administered, i.e., one which is attached to a

10 therapeutic radiolabel. Preferred radiolabels are beta emitting isotopes such as 90 Y or
I31 I, but any radioisotope may be used so long as it may be effectively conjugated to
the antibody, it has a relatively short decay range, and it succeeds in killing nearby
cells, i.e., the cells to which it is targeted. Typically, the radiolabel will be attached by
use of a chelator, e.g., DTPA.

15 A patient preferably will be treated within one week after administration of the

depleting anti-CD20 antibody, so long as they are not severely cytopenic, e.g.,
platelets <1 50,000. If the patient is cytopenic following treatment with the depleting
antibody, recovery should be allowed to occur, e.g. nadir AGC >1000 or platelets
>1 50,000, before radioimmunotherapy. In cases where cell recovery in the peripheral

20 blood and/or bone marrow is permitted to occur, more depleting antibody may be
administered directly before immunotherapy. Such a secondary dosage may be
administered, for example, at about 250 mg/m 2 for about two weeks directly before or
overlapping with radioimmunotherapy.

Dosages of radiolabeled antibodies will also vary depending on the patient, the
. 25 antibody specificity, half-life, radioisotope stability, etc., and of course, the extent of
disease. Radiolabeled anti-CD22 antibodies are typically administered at a dosage of
about 0.001 to 150 mCi/kg, more preferably 0.1 to 50 mCi/kg, still more preferably
0.1 to 30mCi/kg. Another suitable dosage will range from 10 to 30 mCi/kg. The
dosages of radiation can be determined by the ordinary artisan.

30 It should be clear that the treatment methods disclosed herein may be

combined with other known treatment methods such as chemotherapy or radiotherapy.
Bone marrow or peripheral blood stem cells may be harvested from said patient

WO 01/97858

PCIYUS01/18939

subsequent to treatment with anti-CD20 antibody and prior to treatment with said
radiolabeled antibody in order to effect autologous bone marrow or stem cell
transplantation after radiotherapy.

It may also be useful to treat patients with cytokines in order to up-regulate the
5 expression of CD20 or other target protein on the surface of cancerous B cells prior to
administration of the depleting antibody or the radiolabeled antibody. For up-
regulation of CD20, cytokines useful for this purpose are EL-4, GM-CSF and TNF-
alpha. Cytokines may also be administered simultaneously with or prior to or
subsequent to administration of the depleting antibody or radiolabeled antibody in

10 order to stimulate immune effector functions. Cytokines useful for this purpose
include interferon alpha, GM-CSF and G-CSF.

Chemotherapeutic regimens may be used to supplement the therapies
disclosed herein, and may be administered simultaneously with or sequentially in any
order with administration of said radiolabeled antibody. The chemotherapy regimen

1 5 may be selected from the group consisting of CHOP, ICE, Mitozantrone, Cytarabine,
DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La La chemotherapy regime,
ABVD, CEOP, 2-CdA, FLAG & IDA (with or without subsequent G-CSF treatment),
VAD, M & P, C-Weekly, ABCM, MOPP and DHAP. A preferred chemotherapeutic
regimen is CHOP.

20 The methods of the present invention may be used to treat a variety of B cell

lymphomas but are particularly useful wherein said B cell lymphoma is non-
Hodgkin's lymphoma (NHL). RITUXMAB® has already been approved for the
treatment of low-grade-follicular NHL, but the present inventors have surprisingly
found that RITUXIMAB® is also beneficial for the treatment of intermediate- and

25 high-grade NHL, including bulky disease. Accordingly, the lymphomas which are
treatable by the methods of the present invention include low grade/ follicular non-
Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade/
follicular NHL, intermediate grade diffuse NHL, chronic lymphocytic leukemia
(CLL), high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade

30 small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, ATDS-
related lymphoma and Waldenstrom's Macroglobulinemia, so long as such

WO 01/97858

PCT/US01/18939

lymphomas are accompanied by bone marrow involvement which complicates the
availability of radioimmunotherapy.

Also, the present invention maybe used to treat autoimmune diseases.
Examples thereof include glomerulonephritis, Goodpasture's syndrome, necrotizing
5 vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosis, arthritis,
thrombocytopenia purpura, agranulocytosis, autoimmune hemolytic anemias, immune
reactions against foreign antigens, myasthenia gravis, insulin-resistant diabetes, lupus
(SLE and drug-induced lupus). Further, the present invention may be used to treat or
prevent humoral immune responses against transplanted cells, tissues or organs.
1 0 Exemplary treatment conditions will now be illustrated by the following.

EXAMPLE 1

A patient with non-Hodgkin's is initially treated with RITUXAN®. This
initial treatment comprises administration of 375 mg/m 2 RITUXAN® weekly for four
15 weeks.

A week after this RITUXAN® antibody regimen is completed, the patient is
then treated with a 90 Y radiolabeled humanized anti-CD22 antibody (humanized LL2
antibody disclosed in U.S. Patent 5,789,554, to Leung et al, assigned to
hnmunomedics, incorporated by reference in its entire herein.) The patient is treated
20 with a dosage of ^Y-labeled anti-CD22 antibody ranging from 1 0 to 3 0 mCi.

EXAMPLE 2

A patient who is to be transplanted with a kidney is treated prior to transplant
with RITUXAN® at a dosage of 375 mg/m 2 weekly for four weeks in order to deplete
B-cells prior to transplant and thereby reduce the likelihood of a humoral immune
25 response against the transplanted organ.

Concurrent or within a week after RITUXAN® treatment, the subject is
treated with low dosage of 90 Y radiolabeled humanized anti-CD22 monoclonal
antibody, i.e., at a dosage of 10 to 30 mCi. The treated subject is then transplanted
with the kidney by conventional surgical methods. Preferably, the subject will also be
30 treated with anti-CD40L, anti-B7 or other immunosuppressants, e.g., cyclosporin.

WO 01/97858

PCT/US01/18939

WHAT IS CLAIMED IS:

1 . A method of treating a disease or condition wherein suppression and/or
depletion and/or blocking the function of B-cells is therapeutically beneficial,

5 comprising the steps of:

(i) administering a therapeutically effective amount of cold (non-
radiolabeled) anti-CD20 monoclonal antibody having B cell depleting
activity substantially equivalent to Rituxan®; and

(ii) a<toinistering a therapeutically effective amount of a hot (radiolabeled)
10 anti-CD22 antibody or fragment thereof;

wherein said anti-CD20 antibody and said radiolabeled anti-CD22 antibody or
fragment are administered separately or in combination, and in either order.

2. The method of Claim 1, which is used to treat a B-cell malignancy,
15 leukemia or lymphoma.

3. The method of Claim 1, which is used to treat an autoimmune disease.

4. The method of Claim 2, wherein said disease is non-Hodgkins
20 lymphoma.

5. The method of Claim 2, wherein said malignancy, leukemia or
lymphoma is selected from the group consisting of low grade/ follicular non-
Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade/

25 follicular NHL, intermediate grade diffuse NHL, chronic lymphocytic leukemia

(CLL), high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade
small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related
lymphoma and Waldenstrom's Macroglobulinemia.

30 6. The method of Claim 1, wherein the amount of said cold anti-CD20

antibody ranges from 0.1 mg to 500 mg/m 2 per week.

WO 01/97858

PCT/US01/18939

7. The method of Claim 6, wherein the amount of said cold anti-CD20
antibody is at least about 500 mg/m 2 weekly.

8. The method of Claim 7, wherein said dosage is about 375 mg/m 2
5 weekly for four weeks.

9. The method of Claim 1 , wherein said radiolabeled anti-CD22 antibody
or fragment is an yttrium-labeled anti-CD22 antibody or fragment.

10 10. The method of Claim 6, wherein said radiolabeled anti-CD22 antibody

fragment is a Fab 2 , Fab, Fv, or domain deleted antibody.

1 1 . The method of Claim 9, wherein said anti-CD22 antibody is a 90 Y
labeled humanized LL2 antibody.

12. The method of Claim 11, wherein the dosage of said radiolabeled
antibody ranges from 10 to 30 mCi.

1 3 . The method of Claim 1 2, wherein said radiolabeled anti-CD22

20 antibody is administered about a week after an RITUXAN® antibody therapeutic
regimen has been completed.

14. The method of Claim 13, which further includes administration of a
chemotherapeutic agent.

15. The method of Claim 13, which further includes administration of a
cytokine.

16. The method of Claim 1 wherein the anti-CD20 antibody is Rituxan®.

17. The method of Claim 1 6 wherein the condition treated is a B cell
malignancy, leukemia or lymphoma.

WO 01/97858

PCT/US01/18939

18. The method of Claim 5 wherein the anti-CD20 antibody is Rituxan®.

19. The method of Claim 1 6 wherein the disease is transplant.

20. The method of Claim 1 6 wherein the condition is cell therapy.

2 1 . The method of Claim 1 6 wherein the disease is an autoimmune
disease.

22. The method of Claim 2 1 wherein the autoimmune disease is a B cell
related autoimmune disease.

23 . The method of Claim 1 wherein the condition is cell therapy.

24. The method of Claim 1 wherein the condition is allergy.

25. The method of Claim 1 wherein the condition is a treatment involving
the administration of an antigenic moiety.

26. The method of Claim 25 wherein the antigenic moiety is a therapeutic
protein.

27. The method of Claim 1 wherein the disease is a solid tumor wherein B
25 cells promote tumor growth but are not themselves the origin of the tumor.

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual Property Organization
International Bureau

; IIIIIJ'II I Hi'lll.lM M l.li;] 1MIIIIIII llli.llll. 11,11; llll.hllfl

(43) International Publication Date (10) International Publication Number

27 December 2001 (27.12.2001) PCT WO 01/097858 A3

(51) International Patent Classification 7 : A6IK 51/10,
47/48, A6 IP 35/00

(21) International Application Number: PCT/US01/18939

(22) International Filing Date: 14 June 2001 (14.06.2001)

(25) Filing Language: English

(26) Publication Language: English

(30) Priority Data:

60/212,668

20 June 2000 (20.06.2000) US

(71) Applicant: IDEC PHARMACEUTICALS CORPORA-
TION [US/US]; 3030 Callan Road, San Diego, CA 92121
(US).

(72) Inventor: WHITE, Christine; P.O. Box 9242, Rancho
Santa Fe, CA 92067 (US).

(74) Agents: TESKIN, Robin, L. et ai.; Pillsbury Winthrop
LLP, 1600 Tysons Boulevard, McLean, VA 22102 (US).

(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,

CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
GM, MR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK,
SL, TJ, TM, TO, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW.

(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).

Published:

— with international search report

— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments

(88) Date of publication of the international search report:

8 August 2002

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.

00
ITS
00

^ (54) Title: COLD ANTI-CD20 ANTIBODY/RADIOLABELED ANTI-CD22 ANTIBODY COMBINATION

(57) Abstract: Treatment of B-cell associated diseases including autoimmune and B-cell malignancies such as leukemias, lym-
phomas, using the combination of an anti-CD20 antibody, preferably R1TUXAN® and a radiolabeled anti-CD22 antibody, prefer-
ably an '"'Y labeled humanized anti-CD22 antibody, is described. These therapeutic regimens provide for enhanced depletion of B
cells, and therefore reduce the risk in B cell malignancy treatment of relapse associated with RTTUXAN 11 ' and, moreover, provide
for prolonged immunosuppression of B-cell immune responses, especially in the context of autoimmune diseases and transplant.

INTERNATIONAL SEARCH REPORT

In" 1 " lonal Application No

PUT/US 01/18939

A. CLASSIFICATION OF SUBJECT MATTER

IPC 7 A61K51/10 A61K47/48 A61P35/00

According to International Patent Classification (IPC) or to bolti national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system Mowed by classification symbols)

IPC 7 A61K

Documentation searched other than minimum documentation to the extent that such documents are included In the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

CHEM ABS Data, EMBASE , BIOSIS, EPO-Internal

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category • Citation of document, with Indication, where appropriate, of the relevant passages

Relevant to claim No.

WO 98 42378 A (GOLDENBERG DAVID M
;IMMUN0MEDICS INC (US))
1 October 1998 (1998-10-01)
cited in the application
claims 1,9,14,15,18

US 5 789 554 A (HANSEN HANS ET AL)

4 August 1998 (1998-08-04)

cited 1n the application

claims; example 9

WO 00 20864 A (BIOCRYSTAL LTD)
13 April 2000 (2000-04-13)
cited 1n the application

_/--

1-27

X Further documents are listed in the continuation of box C.

Patent family members are listed In annex.

° Special categories of cited documents :

•A" document defining the general slate of the art which Is not
considered to be of particular relevance

'E' earlier document but published on or after the international
filing date

*L" document which may throw doubts on priority claim(s)or
which is cited to establish the publication date of another
citation or other special reason (as specified)

"O" document referring to an oral disclosure, use, exhibition or
other means

"P" document published prior to the international filing date but
later than the priority date claimed

"T" later document published after the International filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention

'X' document of particular relevance; the claimed Invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document Is taken alone

■Y" document of particular relevance; the claimed invention
cannot be considered to Involve an inventive step when the
document Is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
In the art.

'&• document member of the same patent family

Dale of the actual completion of the international search

24 May 2002

Date of mailing of the international search report

03/06/2002

Name and mailing address of the ISA

European Patent Office, P.B. 5816 Patentlaan 2
NL-2280HVRIjSWijk
TeL (+31-70) 340-2040, Tx. 31 651 epo nl,
Fax: (+31-70) 340-3016

Authorized officer

Berte, M

Foim PCT/ISA/210 (second sheet) (July 1892)

page 1 of 2

INTERNATIONAL SEARCH REPORT

lnf~ "onal Application No

PCT/US 01/18939

C(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category •

Citation of document, with Indlcation.where appropriate, of the relevant passages

Relevant lo claim No.

p,x

WO 00 74718 A (HANSEN HANS J ;GOLDENBERG
DAVID M (US); IMMUNOMEDICS INC (US))
14 December 2000 (2000-12-14)
claims 1 , 12-18,21 , 31

1-27

P,Y

WO 01 10461 A (RASTETTER WILLIAM ;WHITE
CHRISTINE (US); IDEC PHARMA CORP (US))
15 February 2001 (2001-02-15)
claims

1-27

P,A

WO 00 67796 A (GENENTECH INC ; IDEC
PHARMACEUTICALS INC (US))
16 November 2000 (2000-11-16)
claims 1,2,9,11

1-27

WO 02 22212 A (IDEC PHARMA CORP)
21 March 2002 (2002-03-21)
claims 1-8

W0 02 04021 A (IDEC PHARMA CORP)
17 January 2002 (2002-01-17)
claims 1-13

P,X

US 6 090 365 A (BUTCHKO GREGORY M ET AL)
18 July 2000 (2000-07-18)
claims 1,11,13

1-27

Form PCT/ISA/210 (continuation of second sheet) (July 1992)

page 2 of 2

IN 1 bKIMA 1 ICJNAL SEARCH REPORT

Information on patent family members

lj tlonal Application No

PTJT/US 01/18939

Patent document
cited in search report

Publication
date

Patent family
member(s)

Publication
date

9842378

01-

•10-

-1998

728325 B2

04-01-2001

All

o/oiuy© a

on in iciqo
tU-lU-iyyo

TP
tr

OOAQfiAA Al

uyoyooo mi

1 9— 01 -9noo

IP
Or

9001 kibosh t

10— 1U— tUU 1

DlOO/Hf 01

oa- 09_9noi

QQ/1927Q Al

u 1— 1U— iyro

O0UOJ7J Ol

0-9001

JOUL'f JO M

04-1 1-1 QQA.

5789554

04-

-08-

-1998

6187287 Bl

13-02-2001

All

1979fiOi; A

u/ uj iyyo

99— (19— 1 QQA

tt Ut 133U

(1771 90S A1
U/ / l&Uo HI

07— OK— 1 QQ7

u/ uo— iyy /

li'jyuy h

9Q— 1 0—1 QQQ

to-iu-iyyy

3053873 B2

19-06-2000

10505231 T

26-05-1998

yoO'ly^b Al

22-02-lyyo

0020864

13-

-04-

-2000

6224866 Bl

01-05-2001

1442700 A

26-04-2000

1 t not a At

1127260 Al

OA AO AAA1

29-08-2001

1 IA

0020864 Al

1 0 Ail onnn

13-04-2000

2001033839 Al

25-10-2001

0074718

14-

-12-

-2000

5600500 A

28-12-2000

1194167 Al

10-04-2002

0074718 Al

14-12-2000

0110461

15-

-02-

-2001

7052100 A

05-03-2001

0110461 Al

15-02-2001

0067796

16-

-11-

-2000

4714300 A

21-11-2000

0011197 A

19-02-2002

1176981 Al

06-02-2002

20015417 A

07-01-2002

0067796 Al

16-11-2000

0222212

21-

-03-

-2002

0222212 A2

21-03-2002

0222687 A2

21-03-2002

0204021

17-

-01-

-2002

2002006404 Al

17-01-2002

0204021 Al

17-01-2002

2002028178 Al

07-03-2002

US 6090365

18-07-2000 US

5595721 A

21-01-1997

6287537 Bl

11-09-2001

5843398 A

01-12-1998

6015542 A

18-01-2000

Form PCT/ISA/210 (patent lamlly annex) (July 1892)

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